Hx Rats Research Articles

Age-Dependent Electrocorticogram Dynamics and Epileptogenic Responsiveness in Rats Subjected to Prenatal Hypoxia

Using electrocorticogram (ECoG) analysis, we compared age-related dynamics of general neuronal activity and convulsive epileptiform responsiveness induced by intracortical microinjections of 4-aminopyridine (4-AP) in control Wistar rats and those subjected to prenatal hypoxia (Hx; E14; 7% O₂, 3 h). The studies were carried out in three age periods roughly corresponding to childhood (P20–27), adolescence (P30–45), and adulthood (P90–120). It was found that in the process of postnatal development of the control rats, the peak of the ECoG power spectrum density (PSD) of the theta rhythm during wakefulness shifted from the low to the higher frequency, while in the Hx rats this shift had the opposite direction. Moreover, the Hx rats had different frequency characteristics of the ECoG PSD and longer episodes of spike-and-wave discharges caused by 4-AP injections compared to the controls. The total ECoG PSD of slow-wave sleep (1–5 Hz) was also dramatically decreased in the process of development of the Hx rats. Such alterations in PSD could be explained by the changes in balance of the excitation and inhibition processes in the cortical networks. Analyzing protein levels of neurotransmitter transporters in the brain structures of the Hx rats, we found that the content of the glutamate transporter EAAT1 was higher in the parietal cortex in all age groups of Hx rats while in the hippocampus it decreased during postnatal development compared to controls. Furthermore, the content of the vesicular acetylcholine transporter in the parietal cortex, and of the inhibitory GABA transporter 1 in the hippocampus, was also affected by prenatal Hx. These data suggest that prenatal Hx results in a shift in the excitatory and inhibitory balance in the rat cortex towards excitation, making the rat’s brain more vulnerable to the effects of proconvulsant drugs and predisposing animals to epileptogenesis during postnatal life.

Impact of chronic free hemoglobin infusion on skeletal muscle microvascular PO2 dynamics in rats

Patients with hemolytic diseases (i.e. sickle cell anemia) exhibit severely compromised exercise capacity and quality of life. This is likely due, at least in part, to chronic exposure to free hemoglobin (Hb) which results in a robust reduction in nitric oxide (NO) bioavailability and, particularly when combined with systemic hypoxia, enacts a proinflammatory cascade that causes vascular dysfunction and remodeling within the pulmonary circulation. Similar downstream effects may occur which could disrupt the QO2/VO2 relationship within skeletal muscle and thus impair tolerance to exercise. Therefore, the purpose of this investigation was to test the hypothesis that chronic exposure to free Hb would lower the microvascular PO2 (PO2mv, the pressure head that facilitates blood‐myocyte O2 flux) at rest and during muscle contractions with a greater deficit observed in rats exposed to hypoxia+Hb. Using programmable micropump technology, 8 young‐adult male Sprague‐Dawley rats were housed in normoxia (Hb+Nx, n=4) or hypoxia (Hb+Hx, n=4) while exposed to 60 mg/day Hb for 18 days while 4 rats were outfitted with dummy infusion pumps (SHAM, n=4) and were housed in normoxia. Following infusion, all rats performed a graded treadmill exercise test to fatigue for the determination of endurance capacity. PO2mv was then measured during 180s of electrically induced (1‐Hz) twitch spinotrapezius muscle contractions. There were no between group differences in exercise tolerance (average run time 16–18 min, p>0.05). While there were no significant differences in any PO2mv kinetic parameter, exposure to hypoxia and Hb resulted in a significantly greater resting baseline (SHAM: 28.3 ± 4, Hb+Nx: 31.5 ± 2, Hb+Hx: 42.8 ± 3 mmHg) and contracting steady‐state PO2 (SHAM: 20.70 ± 4, Hb+Nx: 26 ± 2, Hb:Hx: 36.3 ± 2 mmHg) when compared to SHAM and Hb+Nx conditions, while the contracting steady‐state PO2 was also significantly higher in Hb+Nx vs. SHAM (p<0.05). Blood hematocrit was significantly higher in Hb+Hx when compared to SHAM and Hb+Nx with no significant differences between SHAM and Hb+Nx (SHAM: 48.5 ± 3, Hb+Nx: 43.1 ± 3, Hb+Hx: 54.7 ± 2%, p<0.05). Contrary to our original hypothesis, combined exposure to free Hb and hypoxia resulted in a greater baseline and contracting steady‐state PO2mv while Hb alone raised the steady‐state PO2mv relative to SHAM operated rats. Given that free Hb results in oxidative stress and cellular damage, the elevated PO2mv in both Hb groups likely reflects an impaired ability to utilize oxygen (↓VO2) within the skeletal muscle. This is supported by the lack of differences in exercise tolerance in Hb vs. SHAM despite an increased Hct (i.e. oxygen carrying capacity) observed in the Hb+Hx rats and significantly greater steady‐state PO2mv in both Hb groups. These results carry robust implications for those suffering from hemolytic diseases and provides a potential mechanism by which chronic exposure to free Hb impacts exercise tolerance and quality of life.Support or Funding InformationFunded by NIH (R01HL125642) to DCI and Colorado Nutrition and Obesity Research Center (P30DK048520) award to SKF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased hippocampal brain-derived neurotrophic factor and impaired cognitive function by hypoglossal nerve transection in rats.

The hypoglossal nerve controls tongue movements, and damages of it result in difficulty in mastication and food intake. Mastication has been reported to maintain hippocampus‐dependent cognitive function. This study was conducted to examine the effect of tongue motor loss on the hippocampus‐dependent cognitive function and its underlying mechanism. Male Sprague Dawley rats were subjected to the initial training of Morris water maze task before or after the bilateral transection of hypoglossal nerves (Hx). When the initial training was given before the surgery, the target quadrant dwelling time during the probe test performed at a week after the surgery was significantly reduced in Hx rats relative to sham‐operated controls. When the initial training was given after the surgery, Hx affected the initial and reversal trainings and probe tests. Brain‐derived neurotrophic factor (BDNF) expression, cell numbers and long‐term potentiation (LTP) were examined in the hippocampus on the 10th day, and BrdU and doublecortin staining on the 14th day, after the surgery. Hx decreased the hippocampal BDNF and cells in the CA1/CA3 regions and impaired LTP. BrdU and doublecortin staining was decreased in the dentate gyrus of Hx rats. Results suggest that tongue motor loss impairs hippocampus‐dependent cognitive function, and decreased BDNF expression in the hippocampus may be implicated in its underlying molecular mechanism in relation with decreased neurogenesis/proliferation and impaired LTP.

Increased Activation of M2 Macrophages in 1 Year‐Old Rat Lungs Following Neonatal Hyperoxia Exposure

RationaleMacrophages are activated in response to insult. M1 macrophage polarization is associated with inflammation and tissue deterioration, whereas the M2 macrophage corresponds to wound repair and angiogenesis and has an anti‐inflammatory phenotype. Lipopolysaccharide (LPS) or interferon gamma (IFNγ) promote M1 differentiation and up‐regulate pro‐inflammatory cytokines like interleukin‐ (IL‐) 1β, IL‐6, and IL‐12 whereas M2 differentiation is regulated by IL‐4. Since hyperoxia exposure induces inflammation leading to the pathology seen in bronchopulmonary dysplasia (BPD) through induction of M1 macrophages, we tested the macrophage population in lung tissue of 1 year‐old rats. We postulated an increased activation of M2 macrophages in lungs of hyperoxia‐exposed rats, suggesting a continued wound healing response. Collagen levels were also assessed. We expected to see an increase in collagen levels of hyperoxic rats, suggesting that there is persistent phenotype after one year.MethodsNewborn rats were exposed to 85% hyperoxia (Hx) or normoxia (Nx) (21%) for 14 days and then kept at Nx conditions. At one year, the lung was lavaged with PBS, the left lung was inflated and fixed (4% formaldehyde). Cells were isolated using a 100 μm cell strainer, red blood cells were lysed (ammonium chloride), and the cells frozen in freezing medium. For flow cytometry, the cells were stained for CD45, CD24, CD163, CD80, CD86, CD204, and CD206. Analysis was done with an LSR‐2. Collagen levels were assessed using Masson's trichome‐stained lung sections. A blinded third party divded each image into grids and ranked each square on a scale of 0–3 (0=none, 3=severe) for fibrosis.ResultsAnalysis of CD80 and CD86 showed an increased proportion of the M1 macrophages in the Hx lung compared to the Nx lung (11.7% higher in Hx). The Hx M1 showed more of activation marker CD163 compared to the Nx M1 in the CD24− cell population (12% higher in Hx). Similarly, the proportion of CD204+ M2 was increased within the CD45+ cell population of Hx (6.3% higher in Hx). Comparing M1 to M2 macrophages, Hx exposure shifted the ratio of macrophages toward an M2 response. These findings were supported by the histologic assessment of collagen showing increased lung collagen in Hx compared to Nx rats (p=0.008).ConclusionOne year after neonatal Hx exposure, adult rat lungs demonstrate a macrophage population shift towards M2, suggesting a continued anti‐inflammatory response. Further, the increased level of collagen in Hx rats suggests that there is persistent damage after one year. The data indicates that humans born preterm with BPD may have long term alterations in inflammatory and repair pathways.Support or Funding InformationNHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)

Effect of chronic undernutrition on body mass and mechanical bone quality under normoxic and altitude hypoxic conditions.

Both undernutrition and hypoxia exert a negative influence on both growth pattern and bone mechanical properties in developing rats. The present study explored the effects of chronic food restriction on both variables in growing rats exposed to simulated high-altitude hypoxia. Male rats (n 80) aged 28 d were divided into normoxic (Nx) and hypoxic (Hx) groups. Hx rats were exposed to hypobaric air (380 mmHg) in decompression chambers. At T0, Nx and Hx rats were subdivided into four equal subgroups: normoxic control and hypoxic controls, and normoxic growth-restricted and hypoxic growth-restricted received 80 % of the amount of food consumed freely by their respective controls for a 4-week period. Half of these animals were studied at the end of this period (T4). The remaining rats in each group continued under the same environmental conditions, but food was offered ad libitum to explore the type of catch-up growth during 8 weeks. Structural bone properties (strength and stiffness) were evaluated in the right femur midshaft by the mechanical three-point bending test; geometric properties (length, cross-sectional area, cortical mass, bending cross-sectional moment of inertia) and intrinsic properties of the bone tissue (elastic modulus) were measured or derived from appropriate equations. Bone mineralisation was assessed by ash measurement of the left femur. These data indicate that the growth-retarded effects of diminished food intake, induced either by food restriction or hypoxia-related inhibition of appetite, generated the formation of corresponding smaller bones in which subnormal structural and geometric properties were observed. However, they seemed to be appropriate to the body mass of the animals and suggest, therefore, that the bones were not osteopenic. When food restriction was imposed in Hx rats, the combined effects of both variables were additive, inducing a further reduction of bone mass and bone load-carrying capacity. In all cases, the mechanical properties of the mineralised tissue were unaffected. This and the capacity of the treated bones to undergone complete catch-up growth with full restoration of the biomechanical properties suggest that undernutrition, under either Nx or Hx conditions, does not affect bone behaviour because it remains appropriate to its mechanical functions.

Erythropoietin improves motor and cognitive deficit, axonal pathology, and neuroinflammation in a combined model of diffuse traumatic brain injury and hypoxia, in association with upregulation of the erythropoietin receptor

BackgroundDiffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia.MethodsSprague–Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30 minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-α (5000 IU/kg) or saline at 1 and 24 hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests; 2) axonal pathology (NF-200); 3) callosal degradation (hematoxylin and eosin stain); 3) dendritic loss (MAP2); 4) expression and localisation of the EPO receptor (EpoR); 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1β.ResultsEPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI + Hx). A single dose of EPO at 1 hour reduced axonal damage in the white matter of TAI + Hx rats at 1 day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI + Hx rats; however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1 day after TAI + Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1β to sham levels 2 hours after TAI + Hx, concomitant to a decrease in CD68 positive cells at 7 and 14 days.ConclusionsWhen administered EPO, TAI + Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO’s neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO’s neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.

Growth inhibition and compensation in response to neonatal hypoxia in rats

BackgroundHypoxia is an important disease mechanism in prematurity, childhood asthma and obesity. In children, hypoxia results in chronic inflammation.MethodsWe investigated the effects of hypoxia (Hx) (12% O2) during postnatal day 2 to 20 in rats. Control groups were normoxic (Nc), and normoxic growth restricted (14 pup liters) (Gr).ResultsHypoxia decreased growth similar Gr. Hx increased plasma TNFα and IL-6 and decreased IGF-I and VEGF. Hypoxia resulted in right ventricular (RV) hypertrophy but disproportionate decrements in limb skeletal muscle (SM) growth. miR206 was depressed in the hypertrophied RV of Hx rats while increased in growth retarded SM. Hx resulted in a decreased RV mRNA for myostatin but had no effect on SM myostatin. The mRNA for hypoxia sensitive factors such as HIFα was depressed in the RV of Hx rats suggesting negative feedback.ConclusionThe results indicate that Hx induces a proinflammatory state that depresses growth regulating mechanisms and that tissues critical for survival, such as the heart, can escape from this general regulatory program to sustain life. This study identifies accessible biomarkers for evaluating the impact of interventions designed to mitigate the long-term deleterious consequences of hypoxia that all too often occur in babies born prematurely.

Impact of Postnatal Hypoxia on MicroRNA Expression in Heart and Muscle of Adult Rats

Preterm babies are at risk for lifelong abnormalities in cardiovascular and respiratory systems. The mechanisms and interventions to correct these abnormalities are unknown. Hypoxia is common during the neonatal period in these infants. The acute effects of hypoxia early in life could produce epigenetic programming resulting in long term effects. We studied the acute and long term effects of neonatal hypoxia (Hx, 12% O2) during postnatal days 2 to 20. Acutely, hypoxia severely reduced somatic growth, but produced cardiac right ventricular (RV) hypertrophy that persisted into adulthood (65 days old). The RV of adult Hx rats had higher content of contractile proteins and DNA. At 21 days, the expression of miR‐1 and miR‐206 was significantly downregulated in the RV of Hx rats. The growth of skeletal muscle was disproportionately depressed in Hx rats at 21 days but recovered after 45 days of normoxia. At 21 days the expression of miR‐1 and miR‐206 in skeletal muscle, was upregulated (~2 fold) while miR‐133a was downregulated (~22%). At 65 days of age the expression of miR‐1 remained elevated in the skeletal muscle of male Hx rats. These results indicate that microRNAs participate in reprogramming adaptations to a neonatal hypoxic episode in the heart and skeletal muscles of rats that can persist into adulthood. Supported by NIH Grants P01HD048721 and UL1RR031985.

Repetitive spinal electromagnetic stimulation opens a window of synaptic plasticity in damaged spinal cord: role of NMDA receptors

As we reported previously, propagation of action potentials through surviving axons is impaired dramatically, resulting in reduced transmission to lumbar motoneurons after midthoracic lateral hemisection (HX) in rats. The aim of the present study was to evoke action potentials through the spared fibers using noninvasive electromagnetic stimulation (EMS) over intact T2 vertebrae in an attempt to activate synaptic inputs to lumbar motoneurons and thus to enhance plasticity of spinal neural circuits after HX. We found that EMS was able to activate synaptic inputs to lumbar motoneurons and motor-evoked potentials (MEP) in hindlimb muscles in adult anesthetized rats. Amplitude of MEP was attenuated in parallel with the decline of responses recorded from the motoneuron pool after HX. Repetitive EMS (50 min, 0.2 Hz) facilitated the amplitudes of responses elicited by electric stimulation of lateral white matter or dorsal corticospinal tracts in HX rats. Facilitation sustained for at least 1.5 h after termination of EMS. The N-methyl-d-aspartate (NMDA) receptor blocker MK-801, injected intraspinally close to the recording electrode prior to EMS, did not alter these responses but blocked the EMS-induced facilitation, suggesting that activation of NMDA receptors is required to initiate an EMS-evoked increase. When MK-801 was administered after EMS-induced facilitation was established, it induced depression of these elevated responses. Results suggest that repetitive EMS over intact vertebrae could be used as a therapeutic approach to open a window of synaptic plasticity after incomplete midthoracic injuries, i.e., to activate NMDA receptors in the lumbar motoneuron pool at synaptic inputs and to strengthen transmission in damaged spinal cord.

Rodent estrous cycle response to incomplete spinal cord injury, surgical interventions, and locomotor training.

Estrous cycle disruption after spinal cord injury (SCI) in female rats is a common phenomenon. It remains unknown, however, if the aberrant estrous cycle is a result of an injury to the spinal cord itself or due to the general stress associated with surgical interventions. We addressed this issue by determining estrous cyclicality in female rats after a spinal cord hemisection (HX), implantation of EMG wires into selected hind limb muscles, and/or injections of tracer dyes into the spinal cord. Because it is known that aerobic exercise can enhance the recovery of locomotor function in rodents with an incomplete SCI, we also determined if locomotor training positively impacts the disrupted estrous cycle after an HX. Estrous cycle assessments were made during a 5-8 week period in 27 female rats before and after HX, EMG, and/or dye injection surgeries and in HX rats that recovered spontaneously or underwent locomotor training. Our results show that estrous cyclicality was disrupted (cycle length >5 days) in approximately 76%, 46%, and 50% of the rats after HX, EMG, and dye injection surgeries, respectively. The cyclicality, however, was disrupted for a longer period after HX than after EMG or dye injection surgeries. Furthermore, estrous cycle mean length was shorter in the trained than nontrained HX group. These results suggest that estrous cycle disruption after a major SCI is a consequence of both the direct injury to the spinal cord and to the associated stress. Moreover, moderate aerobic exercise initiated early after a spinal cord HX returns the duration of the estrous cycle toward normal.

Post-Traumatic Hypoxia Exacerbates Brain Tissue Damage: Analysis of Axonal Injury and Glial Responses

Traumatic brain injury (TBI) resulting in poor neurological outcome is predominantly associated with diffuse brain damage and secondary hypoxic insults. Post-traumatic hypoxia is known to exacerbate primary brain injury; however, the underlying pathological mechanisms require further elucidation. Using a rat model of diffuse traumatic axonal injury (TAI) followed by a post-traumatic hypoxic insult, we characterized axonal pathology, macrophage/microglia accumulation, and astrocyte responses over 14 days. Rats underwent TAI alone, TAI followed by 30 min of hypoxia (TAI + Hx), hypoxia alone, or sham-operation (n = 6/group). Systemic hypoxia was induced by ventilating rats with 12% oxygen in nitrogen, resulting in a ∼ 50% reduction in arterial blood oxygen saturation. Brains were assessed for axonal damage, macrophage/microglia accumulation, and astrocyte activation at 1, 7, and 14 days post-treatment. Immunohistochemistry with axonal damage markers (β-amyloid precursor protein [β-APP] and neurofilament) showed strong positive staining in TAI + Hx rats, which was most prominent in the corpus callosum (retraction bulbs 69.8 ± 18.67; swollen axons 14.2 ± 5.25), and brainstem (retraction bulbs 294 ± 118.3; swollen axons 50.3 ± 20.45) at 1 day post-injury. Extensive microglia/macrophage accumulation detected with the CD68 antibody was maximal at 14 days post-injury in the corpus callosum (macrophages 157.5 ± 55.48; microglia 72.71 ± 20.75), and coincided with regions of axonal damage. Astrocytosis assessed with glial fibrillary acidic protein (GFAP) antibody was also abundant in the corpus callosum and maximal at 14 days, with a trend toward an increase in TAI + Hx animals (18.99 ± 2.45 versus 13.56 ± 0.81; p = 0.0617). This study demonstrates for the first time that a hypoxic insult following TAI perpetuates axonal pathology and cellular inflammation, which may account for the poor neurological outcomes seen in TBI patients who experience post-traumatic hypoxia.

Effects of growth hormone on bone modeling and remodeling in hypophysectomized young female rats: a bone histomorphometric study

Growth hormone (GH) deficiency causes decreased bone mineral density and osteoporosis, predisposing to fractures. We investigated the mechanism of action of GH on bone modeling and remodeling in hypophysectomized (HX) female rats. Thirty female Sprague-Dawley rats at age 2 months were divided into three groups with 10 rats each: control (CON) group, HX group, and HX + GH (3 mg/kg daily s.c.) group, for a 4-week study. Hypophysectomy resulted in cessation of bone growth and decrease in cancellous bone mass. Periosteal bone formation decreased and bone turnover rate of endocortical and trabecular surfaces increased as compared to the CON group. GH administration for 4 weeks restored weight gain and bone growth and mitigated decrease in bone density after hypophysectomy. However, trabecular bone mass in the proximal tibial metaphysis remained lower in group HX + GH than in group CON. Dynamic histomorphometric analysis showed that bone modeling of periosteal bone formation and growth plate elongation was significantly higher in group HX + GH than in group HX. New bone formed beneath the growth plate was predominately woven bone in group CON and group HX + GH. Bone remodeling and modeling-remodeling mixed modes in the endocortical and PTM sites were enhanced by GH administration; both bone formation and resorption activities were significantly higher than in group HX. In conclusion, GH administration to HX rats reactivated modeling activities in modeling predominant sites and increased new bone formation. GH administration also increases remodeling activities in remodeling predominant sites, giving limited net gain in the bone mass.

Response of the testis to gonadotrophin replacement in young hypophysectomized vs. gonadotrophin-releasing hormone antagonist-treated rats.

We have studied the response of atrophic Leydig cells to gonadotrophin replacement in young hypophysectomized (HX) and GnRH antagonist (GnRH-ANT)-treated rats. Hypophysectomy was performed at 28 days of age. Age-matched rats were treated with GnRH-ANT from 28 to 51 days of age. From 45 to 51 days of age, animals were injected with 5 IU recFSH, 10 IU hCG or vehicle. Body and testicular weights, as well as the diameter of the seminiferous tubules were significantly higher in GnRH-ANT-treated than in HX rats. Both recombinant FSH and hCG treatments induced a similar increase in testicular weight and tubule diameter in HX and GnRH-ANT-treated rats. However, hCG treatment induced a significantly higher increase in Leydig cell size in HX (3.2-fold) than in GnRH-ANT-treated (1.4-fold) rats. These results suggest that the response of atrophic Leydig cells to gonadotrophin supplementation was partially inhibited in the presence of GnRH antagonist, whereas Sertoli cell-mediated responses seem not to be affected.

Differential Effects of Growth Hormone and Alfa Calcidol on Trabecular and Cortical Bones in Hypophysectomized Rats

Growth hormone (GH) deficiency in children causes severe growth retardation, vitamin D deficiency, and osteopenia. We investigated whether alfacalcidol (1OHD) alone or in combination with GH can improve bone formation. Forty hypophysectomized female rats (HX) at the age of 8 wk were divided into HX, HX + 1OHD (oral 0.25 microg/kg daily), HX+GH (0.666 mg/0.2 mL SC daily) and HX+GH + 1OHD groups for a 4-wk study. Results showed that GH increased body weight, bone area, bone mineral content (BMC), and bone mineral density (BMD), whereas 1OHD only increased BMC and BMD. In cortical bone, GH increased both periosteal and endocortical bone formation resulting in a significant increase in cortical size and area in percentage, whereas 1OHD suppressed endocortical erosion surface per bone surface (ES/BS) without a significant effect on bone formation rate per bone surface (BFR/BS). In trabecular bone, GH mitigated the bone loss by increasing BFR/BS, whereas the 1OHD effect was by suppression of trabecular bone turnover in the HX rats. The combination of GH and 1OHD had no additive effect on increasing trabecular bone mass. In conclusion, GH activates new bone formation and increases bone turnover whereas 1OHD suppresses bone turnover. The combination intervention does not seem to provide any additive benefit.

Long-term exposure to hypobaric hypoxia in rat affects femur cross-sectional geometry and bone tissue material properties

Hypoxia leads to an increase in erythropoiesis, which induces hypertrophy of the erythropoietic marrow and may induce bone resorption. This study investigates the effect of chronic hypobaric hypoxia (simulated high altitude, SHA) on the biomechanics of rat femurs by mechanical tests of diaphyseal stiffness and strength and calculation of some indicators of bone material properties. Adult female rats were exposed to SHA (5500 m, 23.5h/d for 60 d =HX rats; NX=normoxic). This treatment induced reticulocytosis and polycythemia. Bone mineral content did not differ between NX and HX rats at the end of the study. To determine cortical bone mechanical properties, the right femur was assessed using a 3-point bending test. Endosteum and periosteum moved away from the central axis in HX rats as deduced by an increase in the cross-sectional moment of inertia. The two indicators of bone material properties, the elastic modulus and stress at the yielding point, were significantly reduced. However, the strength of the femur as an intact organ ("load capacity" parameters) was not compromised by exposure to SHA. It is thus proposed that the negative effect of SHA on bone material quality was completely offset by an improvement in diaphyseal cross-sectional design, thus allowing a normal biomechanical response to bending of the femur as a whole.

Activation of the hepatic LDL receptor promoter by thyroid hormone

The question of whether mature sterol regulatory element binding protein-2 (SREBP-2) mediates transcriptional activation of the hepatic low density lipoprotein (LDL) receptor by thyroid hormone was investigated. Western blotting analysis and electrophoretic mobility shift assays demonstrated that mature nuclear SREBP-2 protein could be detected in liver nuclear extracts prepared from normal animals but not in extracts prepared from rats rendered hypothyroid either by hypophysectomy (Hx) or thyroidectomy (Tx). Treatment of Hx rats with T 3 restored LDL receptor mRNA levels in about 1 h and caused a 6-fold increase 2.5 h after T 3 administration. However, no detectable mature SREBP-2 was seen in this time period despite a substantial reduction in serum cholesterol levels caused by the T 3 treatment. Deletion of the SRE region from the LDL receptor promoter did not decrease the T 3 response. Thus, the possibility that T 3 may be mediating LDL receptor induction directly via a thyroid response element (TRE) was investigated. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that the rat LDL receptor promoter contains two functional TREs (US-TRE and 2H-TRE). Either one of these elements could support T 3 induction. However, the stronger of these elements is US-TRE at-612 which binds TRβ1 more tightly and when mutated results in a diminished T 3 response. These results indicate that the rapid induction of the hepatic LDL receptor by thyroid hormone is likely due to direct interaction with TREs rather than indirectly by a mechanism involving SREBP-2.

Additive Effect of Vitamin K2 and Risedronate on Long Bone Mass in Hypophysectomized Young Rats

Hypophysectomy (HX) arrests bone growth and induces osteopenia in the long bones of rats. The present study investigated the combined effect of vitamin K(2) and risedronate on long bone mass in HX rats, in order to determine whether treatment with these two agents had an additive effect. Forty female Sprague-Dawley rats were hypophysectomized at 6 weeks of age by the supplier, and were shipped to our laboratory at three days after surgery along with ten intact rats that served as age-matched controls. The study was started on the day when the rats were received. Three HX rats were excluded from the study because of the failure of HX. Forty-seven rats (6 weeks old) were assigned to the following 5 groups by the stratified weight randomization method: intact controls, HX alone, HX + vitamin K(2) (30 mg/kg, p.o., daily), HX + risedronate (2.5 microg/kg, s.c., 5 days a week), and HX + vitamin K(2) + risedronate. The dosing period was 4 weeks. HX resulted in a decrease of the femoral bone area, bone mineral content (BMC) and bone mineral density (BMD), as well as a decrease in the cancellous bone mass of the proximal tibial metaphysis and the total tissue and cortical areas of the tibial diaphysis. These changes were associated with a marked reduction in the serum level of insulin like growth factor (IGF)-I and with elevation of serum alkaline phosphatase (ALP) and pyridinoline. Administration of vitamin K(2) increased the serum ALP level in HX rats, but did not affect any of the other parameters. On the other hand, risedronate ameliorated the decrease of femoral BMD and cancellous bone mass at the proximal tibial metaphysis in HX rats without affecting the serum IGF-I level, as a result of not causing a significant elevation of serum pyridinoline. Vitamin K(2) and risedronate combined had an additive effect on the femoral bone area, BMC and BMD, and the combined treatment group did not show any significant reduction of the total tissue and cortical areas at the tibial diaphysis, as well as a reduced serum pyridinoline level compared with untreated rats and an increased serum ALP level compared with untreated or risedronate-treated rats. These results suggest that risedronate had a positive effect on the BMD and cancellous bone mass of long bones in HX rats. Despite the lack of a significant effect of vitamin K(2) on bone mass parameters, it had an additive effect with risedronate on the BMC, BMD and cortical bone mass of long bones in HX rats.

Effect of vitamin K2 and growth hormone on the long bones in hypophysectomized young rats: a bone histomorphometry study

The purpose of the present study was to determine whether vitamin K(2) and growth hormone (GH) had an additive effect on the long bones in hypophysectomized young rats. Forty-eight female Sprague-Dawley rats (6 weeks old) were assigned to the following five groups by the stratified weight randomization method: intact controls, hypophysectomy (HX) alone, HX + vitamin K(2) (30 mg/kg, p.o., daily), HX + GH (0.625 mg/kg, s.c., 5 days a week), and HX + vitamin K(2) + GH. The duration of the experiment was 4 weeks. HX resulted in a reduction of the cancellous bone volume/total tissue volume (BV/TV) at the proximal tibial metaphysis, as well as decreasing the total tissue area and cortical area of the tibial diaphysis. These changes resulted from a decrease of the longitudinal growth rate and the bone formation rate (BFR)/TV of cancellous bone, as well as a decrease of the periosteal BFR/bone surface (BS) and an increase of endocortical bone turnover (indicated by the BFR/BS) in cortical bone. Administration of vitamin K(2) to HX rats did not affect the cancellous BV/TV or the cortical area. On the other hand, GH completely prevented the decrease of total tissue area and cortical area in cortical bone, as well as the decrease of marrow area and endocortical circumference, by increasing the periosteal BFR/BS compared with that in intact controls and reversing the increase of endocortical bone turnover (BFR/BS). However, GH only partly improved the reduction of the cancellous BV/TV, despite an increase of the longitudinal growth rate and BFR/TV compared with those of intact controls. When administered with GH, vitamin K(2) counteracted the reduction of endocortical bone turnover (BFR/BS) and circumference caused by GH treatment, resulting in no significant difference of marrow area from that in untreated HX rats. These results suggest that, despite the lack of an obvious effect on bone parameters, vitamin K(2) normalizes the size of the marrow cavity during development of the bone marrow in young HX rats treated with GH.

In vivo evaluation of a novel, orally bioavailable, small molecule growth hormone receptor antagonist

IGF-I is regarded as the most sensitive marker of growth hormone (GH) secretion in both GH deficient individuals and in individuals with excessive GH production. Studies on the effect of inhibitors of GH action in normal experimental animals are difficult to evaluate due to the complex relationship and feed back mechanisms of the GH/IGF-I system and the hypothalamo-pituitary axis. To circumvent the GH/IGF-I feedback mechanisms, we have used hypophysectomized (HX) rats treated with GH to assess the potential of a new low molecular weight compound, BVT-A (( N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2-furamide), to act as a GH receptor antagonist in vivo. GH treatment of HX rats induced serum IGF-I, body weight and hepatic mRNA levels of IGF-I, IGFBP-3, ALS and the IGF-I and GH receptors. Co-treatment with BVT-A suppressed all the GH-induced effects. We conclude that the GH substituted HX rat is a useful model for studies on GH receptor antagonists, and for the first time, a small molecule GH receptor antagonist with in vivo activity has been revealed. This opens up for development of new drugs for diseases in which lowering of GH receptor activity would be beneficial.

Synergistic Effect of Vitamin K2 and Prostaglandin E2 on Cancellous Bone Mass in Hypophysectomized Young Rats

Hypophysectomy (HX) results in cessation of bone growth and cancellous osteopenia in rats. It has been reported that prostaglandin E2 (PGE2) improves cortical and cancellous bone mass in HX rats. The purpose of the present study was to examine whether combined administration of vitamin K2 and PGE2 would have a more beneficial effect on bone than single administration of either alone in HX rats. Forty-three female Sprague-Dawley rats, 6 weeks of age, were randomized by the stratified weight method into five groups: intact controls, HX, HX + vitamin K2 (30 mg/kg, p.o., daily), HX + PGE2 (0.83 mg/kg, i.m., 5 days a week), and HX + vitamin K2 + PGE2. The duration of the experiment was 4 weeks. There was a reduction in cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis and a reduction in total tissue area and cortical area (Ct.Ar) of the tibial diaphysis. Vitamin K2 did not affect cancellous BV/TV or Ct.Ar. On the other hand, PGE2 attenuated the loss of cancellous BV/TV in association with higher bone formation rate/bone surface (BFR/BS) and eroded surface (ES)/BS compared with intact controls. PGE2 also increased percent Ct.Ar compared with nontreated HX rats as a result of attenuation of a decrease in periosteal BFR/BS. Vitamin K2 had a synergistic effect with PGE2 on cancellous BV/TV as a result of the suppression of an increase in ES/BS observed by PGE2 treatment. These results suggested that PGE2 had an anabolic action on cancellous and cortical bone and that despite no apparent effect of vitamin K2 on bone, it had a synergistic effect with PGE2 on cancellous bone mass in young HX rats.