Acute minocycline treatment inhibits microglia activation, reduces infarct volume, and has domain-specific effects on post-ischemic stroke cognition in rats.

Ischemic stroke affects millions of individuals worldwide and a high prevalence of survivors experience cognitive deficits. At present, the underlying mechanisms that drive post-stroke cognitive decline are not well understood. Microglia play a critical role in the post-stroke inflammatory response, but experimental studies show that an accumulation of chronically activated microglia can be harmful and associates with cognitive impairment. This study assessed the effect of acute post-stroke minocycline treatment on chronic microglia and astrocyte expression within the infarct and remote white matter regions, as well as its effect on various domains of cognitive function post-stroke. Nine-month-old male rats received an injection of endothelin-1 into the right dorsal striatum to induce transient focal ischemia, and then were treated with minocycline or saline for 4 days post-stroke. Rats were tested using a series of lever-pressing tasks and the Morris water maze to assess striatal-based learning, cognitive flexibility, and spatial learning and reference memory. We found that minocycline-treated rats had smaller stroke-induced infarcts and less microglia activation in the infarct area and remote white matter regions compared to saline-treated rats at 28 days post-stroke. The behavioural testing results differed according to the cognitive domain; whereas minocycline-treated rats trended towards improved striatal-based learning in a lever-pressing task, but cognitive flexibility was unaffected during the subsequent set-shifting task. Furthermore, minocycline treatment unexpectedly impaired spatial learning, yet it did not alter reference memory. Collectively, we show that post-stroke minocycline treatment can reduce chronic microglia activation even in remote brain regions, with domain-specific effects on cognitive function.

Effect of Weight Change on Markers of Bone Turnover and Phosphorus Excretion.

There is little information about how weight change in horses impacts bone turnover and the metabolism of minerals associated with bone. This study evaluated weight change in mature horses as a factor that could alter bone turnover and fecal P output. Fifteen horses (555 ± 8 kg) were assigned to three treatments: weight loss (LO; n = 5), weight maintenance (MA; n = 5), and weight gain (GA; n = 5). Diets contained 75%, 100%, and 145% of maintenance digestible energy requirements for the three treatments, respectively, but contained similar amounts of protein and minerals. At the end of the weight change period (27 ± 6 d), blood samples were analyzed for bone biomarkers and a 5-day total fecal collection was conducted to measure fecal mineral output. Horses fed the MA diet had an average daily weight change that was not different from either the GA or LO treatments, while weight change was different between the GA group and the LO group (0.49 kg/d vs. -1.16 kg/d; P = .017). Weight change was negatively correlated with cross-linking C-terminal telopeptides of type-I collagen, a biomarker of bone resorption (r = -0.62; P = .014) and tended to be positively correlated with bone alkaline phosphatase, a biomarker of bone formation (r = 0.48; P = .068). Also, fecal P output tended to be lower in GA than in LO horses (P = .085), while MA was intermediate and not different, suggesting that weight loss was increasing bone resorption, resulting in a tendency for higher P loss from the body. Weight change in horses can influence bone metabolism as well as mineral excretion.

Effect of subluteal concentrations of progesterone on luteinizing hormone and ovulation in lactating dairy cows.

Two experiments were conducted to determine if administration of progesterone within a low, subluteal range (0.1-1.0 ng/mL) blocks the luteinizing hormone (LH) surge (experiments 1 and 2) and ovulation (experiment 2) in lactating dairy cows. In experiment 1, progesterone was administered to cycling, lactating dairy cows during the luteal phase of the estrous cycle using a controlled internal drug release (CIDR) device. CIDRs were pre-incubated in other cows for either 0 (CIDR-0), 14 (CIDR-14) or 28 days (CIDR-28). One group of cows received no CIDRs and served as controls. One day after CIDR insertion, luteolysis was induced by two injections of prostaglandin (PG) F(2alpha) (25 mg) at 12 h intervals. Two days after the first injection, estradiol cypionate (ECP; 3 mg) was injected to induce a LH surge. Concentrations of progesterone after luteolysis were 0.11, 0.45, 0.78 and 1.20 ng/mL for cows treated with no CIDR, CIDR-28, CIDR-14, and CIDR-0, respectively. LH surges were detected in 4/4 controls, 4/5 CIDR-28, 2/5 CIDR-14 and 0/5 CIDR-0 cows following ECP. In experiment 2, progesterone was administered to cycling, lactating, Holstein cows during the luteal phase of the estrous cycle as in experiment 1. Luteolysis was induced as in experiment 1. The occurrence of an endogenous LH surge and ovulation were monitored for 7 days. Concentrations of progesterone after luteolysis were 0.13, 0.30, 0.70 and 1.20 ng/mL for cows treated with no CIDR, CIDR-28, CIDR-14 and CIDR-0, respectively. LH surges and ovulation were detected in 5/5 controls, 3/7 CIDR-28, 0/5 CIDR-14 and 0/5 CIDR-0 cows. It was concluded that low concentrations of progesterone can reduce the ability of either endogenous or exogenous estradiol to induce a preovulatory surge of LH and ovulation.

Effect of a single injection of progesterone on ovarian follicular cysts in lactating dairy cows.

The objective of this experiment was to evaluate the effect of a single injection of progesterone on the lifespan of ovarian follicular cysts and to examine the fate of follicles that mature following treatment. Lactating Holstein and Jersey cows with ovarian follicular cysts were identified by rectal palpation. The ovaries of cystic cows were then examined by transrectal ultrasonography three times weekly to monitor formation of new follicular cysts. Cows with newly formed follicular cysts were treated either with a single injection of progesterone (200 mg, IM, n = 11) or corn oil vehicle (n = 7). Venous blood samples were collected daily for quantification of progesterone. Blood sampling and ultrasonography continued until ovulation or a new follicular cyst formed. Treatment reduced the lifespan of the cyst by 12 days, from 29.8 +/- 2.3 days in control cows to 17.2 +/- 1.8 days in progesterone-treated cows (P = 0.01). Progesterone treatment also tended to alter the frequency of subsequent follicular events. Ovulation occurred in 4/11 cows that were treated with progesterone whereas none of the vehicle treated cows ovulated (P = 0.07). In conclusion, a single injection of 200mg of progesterone, administered early in the life of an ovarian follicular cyst, shortened its lifespan and in some cases was followed by ovulation of a new follicle.

Effects of acoustic trauma on the auditory system of the rat: The role of microglia.

Exposure to loud, prolonged sounds (acoustic trauma, AT) leads to the death of both inner and outer hair cells (IHCs and OHCs), death of neurons of the spiral ganglion and degeneration of the auditory nerve. The auditory nerve (8cn) projects to the three subdivisions of the cochlear nuclei (CN), the dorsal cochlear nucleus (DC) and the anterior (VCA) and posterior (VCP) subdivisions of the ventral cochlear nucleus (VCN). There is both anatomical and physiological evidence for plastic reorganization in the denervated CN after AT. Anatomical findings show axonal sprouting and synaptogenesis; physiologically there is an increase in spontaneous activity suggesting reorganization of circuitry. The mechanisms underlying this plasticity are not understood. Recent data suggest that activated microglia may have a role in facilitating plastic reorganization in addition to removing trauma-induced debris. In order to investigate the roles of activated microglia in the CN subsequent to AT we exposed animals to bilateral noise sufficient to cause massive hair cell death. We studied four groups of animals at different survival times: 30 days, 60 days, 6 months and 9 months. We used silver staining to examine the time course and pattern of auditory nerve degeneration, and immunohistochemistry to label activated microglia in the denervated CN. We found both degenerating auditory nerve fibers and activated microglia in the CN at 30 and 60 days and 6 months after AT. There was close geographic overlap between the degenerating fibers and activated microglia, consistent with a scavenger role for activated microglia. At the longest survival time, there were still silver-stained fibers but very little staining of activated microglia in overlapping regions. There were, however, activated microglia in the surrounding brainstem and cerebellar white matter.

Effects of free fatty acids on luteinizing hormone and growth hormone secretion in ovariectomized lambs.

The effects of FFA on circulating LH and GH concentrations in ovariectomized ewe lambs were investigated. Lambs (n = 14) were weaned at 2.5 months, ovariectomized at 6.5 months, and used at 8.5 months of age. From weaning until day 0 of the experiment, lambs were fed to maintain body weights (23 kg). On day 0, serum FFA concentrations and mean serum LH concentrations and number and amplitude of LH pulses, as assessed in blood samples collected every 12 min for 4 h, were 6.4 +/- 0.6 mg/100 ml, 0.57 +/- 0.08 ng/ml, 0.45 +/- 0.09 pulses/h, and 0.73 +/- 0.11 ng/ml, respectively. Double the maintenance feeding, beginning day 1, increased (P less than 0.01) body weights by 16% and LH pulse frequency by 82%, but had no effect (P greater than 0.1) on FFA concentrations, mean LH concentrations, or LH pulse amplitude by day 14. On day 14, lambs were infused with lipid (n = 9; 95.8 mg/min) or 0.9% saline solution (n = 5) for 8 h. Blood samples were collected at 12-min intervals for 12 h, beginning 4 h before infusions. FFA levels increased (P less than 0.01) in lipid-infused animals to 27.6 +/- 2.9 mg/100 ml by 4 h of infusion. Mean LH concentrations and LH pulse frequency and amplitude were unaffected (P greater than 0.1) by treatment. In contrast, mean GH concentrations and GH pulse frequency, which were similar (P greater than 0.1) between groups before infusion (14.0 +/- 0.8 ng/ml and 0.36 +/- 0.07 pulses/h, respectively) were decreased by FFA treatment by 51% (P less than 0.01) and 81% (P less than 0.006), respectively. GH pulse amplitude was highly variable and unaffected (P greater than 0.1) by treatment. In summary, elevated FFA levels appear to inhibit the release of GH, but not LH, in the ovariectomized ewe lamb.

Effect of oxytocin on concentrations of prostaglandin H synthase-2 mRNA in ovine endometrial tissue in vivo.

Oxytocin is an acute stimulus of prostaglandin (PG) F2alpha secretion from the ovine uterine endometrium. The high level of PGF2alpha secretion induced by oxytocin and the short half-life of the prostaglandin synthase-2 (PGHS-2) enzyme implies that synthesis of PGHS-2 may be essential at this time. The objective of this study was to determine if the increase in PGF2alpha secretion induced by oxytocin is associated with an increase in PGHS-2 mRNA. In experiment 1, oxytocin induced a rapid increase in serum concentration of 13,14-dihydro-15-keto-prostaglandin F2alpha (the stable metabolite of PGF2alpha; PGFM) that was detected within 7.5 min (P < 0.05) and peaked at 25 min post injection. This was associated with an unusually rapid increase in the concentration of PGHS-2 mRNA at 25 min post oxytocin injection (P < 0.05). Endometrial concentrations of PGHS-2 mRNA returned to basal levels at 90 min post injection. Experiment 2 was conducted to further characterize the time course of induction of PGHS-2 mRNA following oxytocin administration. Oxytocin induced a rapid increase in serum concentrations of PGFM. As in experiment 1, an increase in concentrations of PGHS-2 mRNA was detected at 25 min after oxytocin (P = 0.06). Concentrations of PGHS-2 mRNA were intermediate at 40 min and returned to basal levels at 60 min post injection. Thus, there is a rapid increase in endometrial concentrations of PGHS-2 mRNA following oxytocin stimulation of PGF2alpha secretion. This increase in PGHS-2 mRNA may be required to maintain PGHS-2 enzyme levels during pulsatile secretion of PGF2alpha at luteolysis.

Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F2 alpha in response to oxytocin: role of phospholipase C and diacylglycerol.

The first objective was to describe and evaluate the relationship between the ability of oxytocin to stimulate the activity of phospholipase (PL) C and its ability to stimulate the release of prostaglandin (PG) F2 alpha in ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes after completion of an 11-day steroid replacement protocol. In experiment 1, explants were incubated either in the presence (10(-6) M) or absence of oxytocin for 0, 1, 3, 10, 30 or 100 min to examine the time-course for activation of PLC and release of PGF2 alpha in response to oxytocin. An increase in the activity of PLC was detected at 3 min while an increase in the release of PGF2 alpha was not detected until 10 min (P < 0.05). In experiment 2, explants were incubated in the presence of various oxytocin analogues (10(-6) M) to compare their abilities to activate PLC and release PGF2 alpha. Oxytocin and three receptor agonists stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05) while two oxytocin receptor antagonists had no effect on either response. In experiment 3, explants were incubated in the presence of oxytocin or arginine vasopressin at 10(-9) to 10(-6) M to establish dose-response curves for the activation of PLC and release of PGF2 alpha. For both hormones, significant increases (P < 0.05) in the release of PGF2 alpha were observed at 10(-8) M while increases in PLC activity were not detected until 10(-7) M was used. In experiment 4, explants were pretreated with either U-73122 (an inhibitor of PLC activity) or U-73343 (an inactive analogue of U-73122). Explants were then treated with control medium, oxytocin or AlF4-. Both oxytocin and AlF4-stimulated the activity of PLC and the release of PGF2 alpha (P < 0.05). U-73122 blocked the ability of oxytocin to stimulate the release of PGF2 alpha (P < 0.05) but had no effect on its ability to stimulate the activity of PLC (P > 0.1). Based on the results from these experiments, the role of PLC in mediating the stimulatory effect of oxytocin on the release of PGF2 alpha remains unclear. The second objective was to evaluate the role of diacylglycerol (DAG) in mediating the stimulatory effect of oxytocin on endometrial secretion of PGF2 alpha. In experiment 5, explants were incubated in vitro with varying doses of two DAG analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of progesterone withdrawal on uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes.

Two experiments were conducted to determine if withdrawal of progesterone during the luteal phase of the oestrous cycle affected the ability of the ovine uterus to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin. In Experiment 1, 18 ewes were ovariectomized on Day 9 and Day 12 after oestrus. Ewes were subdivided into three treatment groups (n = 6 per group): Group-1 ewes underwent sham surgery; Group-2 ewes received oestradiol (OVX + O); and Group-3 ewes received oestradiol + progesterone (OVX + O,P). Oxytocin was administered to each ewe on Days 10, 13 and 15 after oestrus. Concentrations of 13, 14-dihydro-15-keto-PGF2 alpha (PGFM) were determined in samples of jugular venous blood for 2 h after oxytocin challenge. The magnitude of the PGFM response 24 h after ovariectomy was greater (P < 0.1) in ewes from which progesterone had been withdrawn (OVX + O) than in ewes in which progesterone was maintained (intact controls and OVX + O,P). Therefore, progesterone appears to exert an inhibitory effect on uterine secretory responsiveness to oxytocin which is removed by progesterone withdrawal. In Experiment 2, ewes were ovariectomized on Day 11 and assigned to 1 of 4 treatment groups (n = 6 per group): Group 1, no steroid replacement (OVX); Group 2, oestradiol replacement (OVX + O); Group 3, progesterone replacement (OVX + P); or Group 4, progesterone + oestradiol replacement (OVX + O,P). Ewes received oxytocin on Day 12 and Day 15. On Day 12, uterine secretory responsiveness to oxytocin was greatest in ewes in the OVX + O group (P < 0.1). Responsiveness was low in ewes in the OVX group, as it was in ewes in both groups that received progesterone replacement. Therefore, the increase in uterine secretory responsiveness to oxytocin following progesterone withdrawal is dependent on oestradiol replacement.

Microwave diathermy treatment for primary dysmenorrhea.

This case report documents the use of microwave diathermy in a 31-year-old woman who had had primary dysmenorrhea since menarche began at age 13 years. For 18 years, she had severe monthly pain, frequently resulting in emergency department admissions and 1 to 3 days lost from work. Conventional treatments, including pain-relieving drugs, anti-inflammatory drugs, muscle relaxants, superficial heat, and oral contraceptives, had all been unsuccessful in relieving or abating the intense and debilitating pain. Microwave diathermy (45 W total power) was administered for 20 minutes each month on the day symptoms began (usually the first day of menstruation). Over a 7-month interval, diathermy was followed by almost-immediate and long-lasting relief of symptoms. During the 7 months of treatment, the patient lost no workdays due to severe pain. This case demonstrates the potential use of microwave diathermy as an effective treatment for women with this condition.

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2 alpha synthesis in bovine endometrium: role of calcium.

The objective of these experiments was to determine the role of Ca2+ during oxytocin-stimulated prostaglandin (PG) F2 alpha release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF2 alpha release. A23,187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF 2 alpha in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca2+ in the cytoplasm by inhibiting endoplasmic reticulum Ca2+/ATPase pumps) stimulated release of PGF2 alpha in a concentration-dependent manner as well (P < 0.13). Oxytocin (10(-6) M), AIF4- (a nonspecific activator of G-proteins; 10(-5) M), A23,187 (10(-5) M), and melittin (a stimulator of phospholipase A2; 10(-4) M) stimulated PGF2 alpha release when explants were incubated in Ca(2+)-free medium (P < 0.10); however, oxytocin, A23,187, or melittin were unable to stimulate PGF2 alpha release when explants were incubated in Ca(2+)-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AIF4- from stimulating phospholipase C activity (P < 0.08). CoCl2 (a nonspecific Ca2+ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca2+ channel blocker) prevented oxytocin from stimulating PGF2 alpha release (P < 0.05). Our results suggest that both extracellular and intracellular Ca2+ may be required for oxytocin to stimulate PGF2 alpha secretion in bovine endometrial tissue.

Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo.

The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P < 0.01). However, endometrial concentrations of cPLA2 mRNA did not change following oxytocin administration (P > 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P < 0.01). Endometrial concentrations of cPLA2 protein were greater in the cytosolic than in the microsomal fraction (P < 0.01). Oxytocin did not affect the amount of cPLA2 protein in either fraction (P > 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

Cellular mechanisms by which oxytocin stimulates uterine PGF2 alpha synthesis in bovine endometrium: roles of phospholipases C and A2.

The objective of these experiments was to identify the cellular mechanisms by which oxytocin stimulates prostaglandin (PG) F2 alpha synthesis in bovine endometrial tissue. Uteri were collected on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to assess PGF2 alpha release or phospholipase (PL) C activity. Oxytocin (10(-6) M) stimulated PGF2 alpha release and PLC activity within 30 min of incubation (P < 0.01). The highest stimulation was observed at 100 min (P < 0.01). Oxytocin stimulated PLC activity at 10(-9) M and higher doses, whereas an increase in PGF2 alpha release was not detected until 10(-8) M (P < 0.09). Melittin, a stimulator of PLA2 activity, stimulated PGF2 alpha release at 10(-6) M and higher doses (P < 0.01). Aristolochic acid, an inhibitor of PLA2 activity, blocked the ability of oxytocin to stimulate PGF2 alpha release at 10(-5) M and higher doses (P < 0.01). Aristolochic acid (10(-4) M) reduced the stimulation of PGF2 alpha release induced by A1F4-, a nonspecific stimulator of G protein (10(-5) M) and melittin (10(-4) M; P < 0.05). Aristolochic acid had no effect on the ability of oxytocin or A1F4- to stimulate PLC activity (P > 0.10). By comparing the time course of stimulation and dose-response relationships between PGF2 alpha and PLC activity, it appears that oxytocin may stimulate PGF2 alpha secretion by activating PLC. The effects of melittin and aristolochic acid indicate that PLA2 may play a role in mediating the stimulatory effect of oxytocin on PGF2 alpha secretion, as well.

Effects of testosterone and 5alpha-dihydrotestosterone on luteal lifespan in dairy heifers.

Endogenous concentrations of testosterone increase approximately 7 d prior to estrus in cattle and goats. Inhibition of testosterone synthesis results in a delay of luteal regression in both species. The purpose of this experiment was to determine if treatment with testosterone or 5alpha-dihydrotestosterone (DHT), 2 to 6 d prior to the endogenous rise in testosterone, would result in premature luteal regression. Sixteen heifers were randomly assigned to one of three treatment groups: 1) Control (n = 6); 2) testosterone (100 mug, n = 5); or 3) DHT (100 mug, n = 5). Each heifer received a single injection of the appropriate steriod on Day 8, 9, 10, 11 or 12 post estrus. Jugular venous blood samples were collected at frequent intervals for 24 h to quantify testosterone, and then daily for 14 d to quantify progesterone. Concentrations of testosterone increased within 15 min of injection of testosterone, and reached a maximum at 30 min. Concentrations were maintained at > 2 ng/ml throughout the first 24 h after injection. Based on concentrations of progesterone, neither androgen had any effect on the lifespan of the corpus luteum or the level of luteal function.

Review of salicylate-induced hearing loss, neurotoxicity, tinnitus and neuropathophysiology.

Salicylate's ototoxic properties have been well established, inducing tinnitus and a sensory hearing loss when administered in high doses. Peripherally, acute dosing of salicylate causes frequency dependent reductions in DPOAEs and CAP amplitudes in low (<10 kHz) and high (>20 kHz) frequencies more than mid frequencies (10-20 kHz), which interestingly corresponds to the pitch of behaviourally-matched salicylate-induced tinnitus. Chronic salicylate dosing affects the peripheral system by causing a compensatory temporary enhancement in DPOAE amplitudes and up-regulation of prestin mRNA and protein expression. Despite salicylate's antioxidant properties, cultured cochlea studies indicate it also impairs spiral ganglion neurons (SGNs) by paradoxically causing an upsurge of superoxide radicals leading to apoptosis. Centrally, salicylate alters γ-aminobutyric acid (GABA) and serotonin mediated neurotransmission in the central nervous system (CNS), which results in classical and non-classical auditory regions showing hyperactivity after salicylate administration. In the auditory cortex (AC) and lateral amygdala (LA), neuron characteristic frequencies (CF) shift upward and downward to mid frequencies (10-20 kHz) altering tonotopy following salicylate administration. Additionally, current source density (CSD) analysis showed enhanced current flow into the supergranular layer of the auditory cortex after a high systemic dose of salicylate. In humans, auditory perception changes following salicylate or aspirin, including decreased word discrimination and temporal integration ability. The results of previous studies have partially identified the mechanisms that are involved in salicylate-induced tinnitus and hearing loss, however to date some interactions remain convoluted. This review discusses current knowledge of salicylate ototoxicity and interactions.

Dissociation of doublecortin expression and neurogenesis in unipolar brush cells in the vestibulocerebellum and dorsal cochlear nucleus of the adult rat.

We have previously shown expression of the protein doublecortin (DCX) in unipolar brush cells (UBCs) in the dorsal cochlear nucleus and vestibulocerebellum of the adult rat. We also saw DCX-immunoreactive elements with the appearance of neuroblasts around the fourth ventricle. Expression of DCX is seen in newborn and migrating neurons and hence considered a correlate of neurogenesis. There were two interpretations of the expression of DCX in UBCs. One possibility is that there might be adult neurogenesis of this cell population. Adult neurogenesis is now well-established, but only for the dentate gyrus of the hippocampus and the subventricular zone. The other possibility is that there is prolonged expression of DCX in adult UBCs that may signal a unique role in plasticity of these neurons. We tested the neurogenesis hypothesis by systemic injections of bromodeoxyuridine (BrdU), a thymidine analog, followed by immunohistochemistry to examine the numbers and locations of dividing cells. We used several different injection paradigms, varying the dose of BrdU, the number of injections and the survival time to assess the possibility of neuronal birth and migration. We saw BrdU-labeled cells in the cerebellum and brainstem; cell division in these regions was confirmed by immunohistochemistry for the protein Ki67. However, neither the numbers nor the distribution of labeled nuclei support the idea of adult neurogenesis and migration of UBCs. The function of DCX expression in UBC's in the adult remains to be understood.

Digestive capacity in weanling and mature horses.

The ability of young and mature horses to digest DM, OM, and NDF was compared using 6 weanling colts and 6 mature (13.2 ± 3.0 yr) geldings. Each colt was paired with a gelding, and the pair was adapted to a diet containing 67% alfalfa cubes and 33% concentrate for 21 d. During the adaptation period, horses were accustomed to housing and all handling procedures. The adaptation period was also used to adjust the amount of feed offered to minimize orts and to maintain similar rates of intake within a pair. After the adaptation period, a 5-d fecal collection period using fecal collection harnesses ensued. The average age of the weanling colts at the start of the 5-d collection period was 181.8 ± 2.9 d. On the morning of the first collection day, Co-EDTA (9 mg Co/kg BW(0.75)) and ytterbium-labeled hay fiber (9 mg Yb/kg BW(0.75)) were added to the concentrate portion of the diet, and horses were closely observed for complete consumption of the markers before additional feed was offered. The fecal collection bags were emptied every 1 to 2 h, and each collection was weighed and subsampled for later measurement of Co and Yb concentrations, which were used to determine the mean retention time (MRT) of the fluid and particulate phases of digesta, respectively. The remaining feces for each horse were composited each day and then subsampled for measurement of DM digestibility (DMD), NDF digestibility (NDFD), and OM digestibility (OMD). During the fecal collection period, DMI was similar between colts and geldings (91.4 and 91.2 g/kg BW(0.75), respectively). There were no differences between colts and mature geldings for DMD, OMD, or NDFD. Across both ages, the MRT of the particulate phase was 24.9 h compared with 21.8 h for the fluid phase (P = 0.002). However, MRT for the particulate phase was not different between colts and mature geldings (24.7 and 25.2 h, respectively). There was no difference in the MRT for the fluid phase between colts and mature geldings (21.5 and 22.0 h, respectively). The results indicated that the digestibility of DM, OM, and NDF in a diet consisting of good-quality cubed forage and concentrate is similar for weanling colts and mature geldings.

Expression of doublecortin, a neuronal migration protein, in unipolar brush cells of the vestibulocerebellum and dorsal cochlear nucleus of the adult rat.

Doublecortin (DCX) is a microtubule-associated protein that is critical for neuronal migration and the development of the cerebral cortex. In the adult, it is expressed in newborn neurons in the subventricular and subgranular zones, but not in the mature neurons of the cerebral cortex. By contrast, neurogenesis and neuronal migration of cells in the cerebellum continue into early postnatal life; migration of one class of cerebellar interneuron, unipolar brush cells (UBCs), may continue into adulthood. To explore the possibility of continued neuronal migration in the adult cerebellum, closely spaced sections through the brainstem and cerebellum of adult (3-16 months old) Sprague-Dawley rats were immunolabeled for DCX. Neurons immunoreactive (ir) to DCX were present in the granular cell layer of the vestibulocerebellum, most densely in the transition zone (tz), the region between the flocculus (FL) and ventral paraflocculus (PFL), as well as in the dorsal cochlear nucleus (DCN). These DCX-ir cells had the morphological appearance of UBCs with oval somata and a single dendrite ending in a brush. There were many examples of colocalization of DCX with Eps8 or calretinin, UBC markers. We also identified DCX-ir elements along the fourth ventricle and its lateral recess that had labeled somata but lacked the dendritic structure characteristic of UBCs. Labeled UBCs were seen in nearby white matter. These results suggest that there may be continued neurogenesis and/or migration of UBCs in the adult. Another possibility is that UBCs maintain DCX expression even after migration and maturation, reflecting a role of DCX in adult neuronal plasticity in addition to a developmental role in migration.

Differential regulation of estrous behavior and luteinizing hormone secretion by estradiol-17ß in ovariectomized dairy cows.

Holstein cows (n = 9) were used in an experiment to characterize the behavioral and endocrine responses to estradiol-17ß when administered at rates designed to maintain peripheral concentrations within a physiological range. Cows were pretreated with progesterone for 3 d. Three days after progesterone treatment was completed, each cow was assigned to one of five estradiol-17ß treatment groups (Doses 0 to 4), calculated to produce and maintain 0, 3, 6, 9, or 12 pg/mL in peripheral blood for 8 h. The experiment was conducted in eight replicates (with 3 to 7 cows each), with no dose repeated in any replicate. In each replicate, at least one additional cow was given an injection of estradiol-17ß (500 µg im, in a corn oil vehicle) to facilitate estrus detection. Estrus was detected by visual observation for 30 min at 4 h intervals. Estrus was defined as a cow that stood to be mounted at least twice during the 50 h interval over which estrus was observed. Jugular venous blood samples were collected at 2 h intervals throughout the infusion and observation periods for quantification of luteinizing hormone (LH). Cows that received the highest dose (Dose 4, n = 7) all showed estrus, whereas those that received the two lowest doses (Dose 0, n = 5; Dose 1, n = 6) did not. Over the course of the experiment, five cows received each dose at least once. Of these, three showed estrus at Doses 2, 3, and 4, whereas the other two showed estrus only at Dose 4. Therefore, individual cows differed in the amount of estradiol-17ß needed to induce estrus. There was a linear effect of dose on duration of estrus (P < 0.01). Estrus was shorter for Dose 2 (8.0 h) than for Dose 4 (18.4 h). The onset of estrus (after start of infusion) tended to be later for Dose 2 (20.0 h) than for Doses 3 and 4 (14.0 and 13.4 h, respectively; P = 0.15). Preovulatory-like surges of LH were induced in all cows at Doses 2, 3, and 4. Surges also were detected in 3 of 5 cows receiving Dose 1. The magnitude of the LH surge was less for Doses 1, 2, and 3 than for Dose 4 (P = 0.06). In contrast to the timing of estrus, the timing of the LH surge (after start of infusion) was not different among doses (P = 0.88). Thus, the hypothalamic centers responsible for regulating expression of estrus and secretion of LH responded differently to estradiol-17ß.

Comparison of in vitro digestibility estimates using the DaisyII incubator with in vivo digestibility estimates in horses.

The objective of this study was to determine if in vitro methodologies developed for the Ankom Daisy(II) incubator could produce accurate estimates of in vivo equine DM digestibility (DMD) and NDF digestibility (NDFD) when equine feces were used as the inoculum source. Four mature geldings were utilized in a 4 × 4 Latin square design experiment with a 2 × 2 factorial arrangement of dietary treatments (timothy hay, alfalfa hay, timothy hay plus oats, and alfalfa hay plus oats), in which the geldings were individually housed and fed. During each 5-d total fecal collection period, feces were collected and composited daily and used to calculate in vivo digestibility. Digestion of the 4 treatment diets was evaluated in vitro using the Daisy(II) incubator. Each incubation vessel of the Daisy(II) was assigned to 1 of the horses and contained 18 filter bags (6 containing the assigned treatment hay, 6 containing hay-oat mix, and 6 containing oats). Three incubation periods were evaluated: 30, 48, and 72 h. Although the 30- and 48-h in vitro estimates were consistently less than the in vivo estimates, they ranked diets in the same order as the in vivo method. For the alfalfa oat diet, timothy diet, and the timothy oat diet, the mean 72-h in vitro DMD and in vivo DMD were not different (P = 0.1444). However, for the alfalfa diet, the DMD estimate from 72-h in vitro incubation was less than the in vivo estimate (P < 0.010). For NDFD, the timothy diet was the only diet, in which the mean 72-h in vitro NDFD estimate was not different than the in vivo estimate. However, the in vitro method correctly ranked the alfalfa-based diets as having greater NDFD estimates than the timothy-based diets. Of the 3 incubation periods, the 72-h period provided digestibility estimates most similar to the in vivo data. Using the methodologies described in this research, the Daisy(II) incubator and equine feces can be used to estimate in vivo DMD of horse feeds.