[Apatinib Suppressed Macrophage-Mediated Malignant Behavior of Hepatocellular Carcinoma Cells via Modulation of VEGFR2/STAT3/PD-L1 Signaling].

Hepatocellular carcinoma (HCC) is the most frequently diagnosed primary liver tumor worldwide. Tumor-associated macrophages (TAMs) usually have a similar phenotype to M2-like macrophages and can participate in tumor progression by secreting cytokines to suppress the immune response and activity of tumor-infiltrating lymphocytes. We investigated the role of M2 macrophages in HCC progression and explored the effects of vascular endothelial growth factor receptor 2 inhibitor-apatinib. As a cellular model of HCC, Hepb3 cell line was used. M2 macrophages were obtained by differentiation of THP-1 cells. The Transwell chamber was used to co-culture M2 macrophages and Hepb3 cells. CCK-8 and EdU assays were conducted to measure cell viability and proliferation capacity. Transwell migration assay was performed to estimate cellular metastatic potential. Cytokine expression levels were assessed by ELISA. Western blotting was used to characterize activation of the VEGFR2/STAT3/PD-L1 axis. It has been shown that co-culture with M2 macrophages increased viability, cytokine production, promoted proliferation, invasion, and migration of Hepb3 cells. The secretion of TGF-ß1, IL-6, MMP-9, and VEGF was significantly increased after co-culture. In contrast apatinib suppressed M2 macrophage-induced proliferation, cell viability, invasion, and migration of Hepb3 cells. Moreover, apatinib markedly decreased expression levels of p-VEGFR2, p-STAT3, and PD-L1 in Hepb3 cells under the co-culture conditions. In conclusion, apatinib treatment can suppress TAMs-mediated malignant behavior of HCC cells via modulation of the VEGFR2/STAT3/PD-L1 signaling pathway.

[Investigation and analysis of airborne allergenic pollen in 4 districts and 5 counties of Hohhot City].

Objective: To investigate the species, concentration and seasonal trends of main airborne allergenic pollen in 4 districts and 5 counties of Hohhot City. Methods: The Department of allergy, Beijing Shijitan Hospital Affiliated to Capital Medical University conducted a cross-sectional study about monitoring the airborne allergenic pollen from August 1, 2021 to July 31, 2022 by the gravitational method in 4 districts and 5 counties of Hohhot City, which include Yuquan District, Xincheng District, Huimin District, Saihan District, Tuoketuo County, Helingeer County, Tumotezuoqi County, Wuchuan County and Qingshuihe County. Daily pollens were counted and identified by optical microscopy, and the data were analyzed. Results: The airborne allergenic pollen was collected every month all year round in 4 districts and 5 counties of Hohhot city. Through the whole year of the total quantity of pollens ranged from 24 850 to 50 154 grains per 1 000 mm2 and two peaks of pollen concentration in air were observed,which happened in spring (from March to May) and in summer and autumn (from July to September). In spring, the main pollens were tree pollens, which principally distributed in Populus pollen (18.29%), Ulmus pollen (8.36%), Pinus pollen (6.20%), Cupressaceae pollen (5.23%), Betulaceae pollen (2.73%), Salix pollen (1.80%) and Quercus pollen (1.16%). In summer and autumn, the main pollens were weed pollens, which mainly included Artemisia pollen (42.73%), Chenopodiaceae pollen or Amaranthaceae pollen (7.46%), Poaceae pollen (2.26%), Humulus pollen or Cannabis pollen (0.60%). Conclusion: There were two peaks of main airborne allergenic pollen in 4 districts and 5 counties of Hohhot City. In the spring peak of pollen, the main airborne pollens were tree pollens. In the summer and autumn peak of pollen, the main airborne pollens were weed pollens. The Artemisia pollen was the most major airborne pollen in this area.

The cAMP responsive element-binding (CREB)-1 gene increases risk of major psychiatric disorders.

Bipolar disorder (BPD), schizophrenia (SCZ) and unipolar major depressive disorder (MDD) are primary psychiatric disorders sharing substantial genetic risk factors. We previously reported that two single-nucleotide polymorphisms (SNPs) rs2709370 and rs6785 in the cAMP responsive element-binding (CREB)-1 gene (CREB1) were associated with the risk of BPD and abnormal hippocampal function in populations of European ancestry. In the present study, we further expanded our analyses of rs2709370 and rs6785 in multiple BPD, SCZ and MDD data sets, including the published Psychiatric Genomics Consortium (PGC) genome-wide association study, the samples used in our previous CREB1 study, and six additional cohorts (three new BPD samples, two new SCZ samples and one new MDD sample). Although the associations of both CREB1 SNPs with each illness were not replicated in the new cohorts (BPD analysis in 871 cases and 1089 controls (rs2709370, P=0.0611; rs6785, P=0.0544); SCZ analysis in 1273 cases and 1072 controls (rs2709370, P=0.230; rs6785, P=0.661); and MDD analysis in 129 cases and 100 controls (rs2709370, P=0.114; rs6785, P=0.188)), an overall meta-analysis of all included samples suggested that both SNPs were significantly associated with increased risk of BPD (11 105 cases and 51 331 controls; rs2709370, P=2.33 × 10-4; rs6785, P=6.33 × 10-5), SCZ (34 913 cases and 44 528 controls; rs2709370, P=3.96 × 10-5; rs6785, P=2.44 × 10-5) and MDD (9369 cases and 9619 controls; rs2709370, P=0.0144; rs6785, P=0.0314), with the same direction of allelic effects across diagnostic categories. We then examined the impact of diagnostic status on CREB1 mRNA expression using data obtained from independent brain tissue samples, and observed that the mRNA expression of CREB1 was significantly downregulated in psychiatric patients compared with healthy controls. The protein-protein interaction analyses showed that the protein encoded by CREB1 directly interacted with several risk genes of psychiatric disorders identified by GWAS. In conclusion, the current study suggests that CREB1 might be a common risk gene for major psychiatric disorders, and further investigations are necessary.

The protocadherin 17 gene affects cognition, personality, amygdala structure and function, synapse development and risk of major mood disorders.

Major mood disorders, which primarily include bipolar disorder and major depressive disorder, are the leading cause of disability worldwide and pose a major challenge in identifying robust risk genes. Here, we present data from independent large-scale clinical data sets (including 29 557 cases and 32 056 controls) revealing brain expressed protocadherin 17 (PCDH17) as a susceptibility gene for major mood disorders. Single-nucleotide polymorphisms (SNPs) spanning the PCDH17 region are significantly associated with major mood disorders; subjects carrying the risk allele showed impaired cognitive abilities, increased vulnerable personality features, decreased amygdala volume and altered amygdala function as compared with non-carriers. The risk allele predicted higher transcriptional levels of PCDH17 mRNA in postmortem brain samples, which is consistent with increased gene expression in patients with bipolar disorder compared with healthy subjects. Further, overexpression of PCDH17 in primary cortical neurons revealed significantly decreased spine density and abnormal dendritic morphology compared with control groups, which again is consistent with the clinical observations of reduced numbers of dendritic spines in the brains of patients with major mood disorders. Given that synaptic spines are dynamic structures which regulate neuronal plasticity and have crucial roles in myriad brain functions, this study reveals a potential underlying biological mechanism of a novel risk gene for major mood disorders involved in synaptic function and related intermediate phenotypes.

[Influencing factors of visual hallucinations in patients with Parkinson's disease and its relationship with sleep disorders].

OBJECTIVE: To investigate the prevalence and influencing factors of visual hallucinations in patients with Parkinson's disease(PD), and to analyze the relationship between visual hallucinations and sleep disorders. METHODS: We recruited 187 patients with PD(H-Y Ⅰ-Ⅲ) from outpatient department in Beijing Hospital. The patients were investigated for general information and the use of medicine. The patients were divided into visual hallucination(VH) group and non-hallucination(non-VH) group. A comparison study was conducted between two groups. We investigated the sleep disorders of PD patients according to Non Motor Symptom Quest(NMSquest) and Parkinson's disease sleep scale(PDSS). Logistic stepwise multiple regression procedures were used to determine the best predictive model of visual hallucinations in patients with PD. RESULTS: (1) 42 cases(22.5%) of PD patients were accompanied by visual hallucinations; (2) the VH group and non-VH group had no difference in age, sex, duration of illness, the scores of Minimum Mental State Examination(MMSE) and levodopa equivalent doses (LED). The scores of Unified Parkinson's Disease Rating Scale(UPDRS) Ⅰ, the Hamilton Rating Scale for Anxiety(HAMA) and the Hamilton Rating Scale for Depression(HAMD) in VH group were significantly higher than those in non-VH group[3.5(2, 5) vs 2 (1, 3); 10(6.75, 15) vs 8(5, 11); 11(7.75, 17) vs 9(5, 13); P<0.05]; (3) the incidences of vivid dreams and REM sleep behavior disorder(RBD) in VH group were significantly higher than those in non-VH group(61.9% vs 40.7%, 71.4% vs 47.6%, P<0.05). There were no significant differences in incidences of excessive daytime sleepiness and restless legs between two groups(P>0.05). The score of PDSS in VH group was significantly lower than that in non-VH group[111(92.75, 128.25) vs 123(109, 135), P<0.05]; (4) the Logistic stepwise multiple regression revealed that vivid dreams(P=0.045) and the score of PDSS(P=0.006) were the independent influencing factors for VH in PD patients. CONCLUSIONS: The incidence of VH in PD with H-Y staging Ⅰ-Ⅱ is 22.5%. The presence of vivid dreams and severe sleep disorder are independently associated with VH in PD.

[Application of low dose CT angiography with 70 kV in patients with peripheral arterial diseases].

Objective: To assess the image quality, radiation dose and diagnostic efficiency of peripheral arterial CT angiography (CTA) performed at tube voltage of 70 and 120 kV. Methods: Between January 2014 and December 2015, a total of 200 consecutive patients with known or suspected lower extremity arteriosclerosis obliterans (LEASO) underwent CTA.Patients were randomly divided into 2 groups by different scanning protocols.Group A (n=100): 70 kV and 0.8 ml/kg contrast agent, group B (n=100): 120 kV and 100 ml contrast agent.The vessel enhancement, image noise, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of 3 segments were quantified for each protocol.30 patients in group A (420 vessels) and 28 patients in group B (384 vessels) confirmed by DSA.Based on vessel assessments, compared with DSA findings, the diagnostic efficacy of 70 kV and 120 kV protocols for the detection of stenoses over 50% was evaluated.The dose of radiation and contrast agent were recorded.Subjective image quality was evaluated. Results: The subjective image quality of segment crural of group A was significantly higher than that of group B (2.20±0.36 vs 1.72±0.34, P<0.01). The enhancement of 3 segments in group A (500 HU) were significantly higher than these in group B (310 HU) (P<0.05). For the detection of stenoses over 50%, the sensitivity, positive and negative predictive values and accuracy of segment crural in group A (98.6%, 95.8%, 98.1%, 96.7%) were significantly higher than that in group B (90.9%, 88.5%, 91.0%, 89.7%) (P<0.05). Mean DLP for 70-kV protocol was significantly lower than that for 120-kV protocol ( (396±34) vs (1 041±159) mGy·cm, P=0.001). Mean dose of contrast agent and the total amount of iodine for 70-kV protocol were significantly lower than that for 120-kV protocol (53.5 vs 100 ml; 18.7 vs 35 g; P<0.01). Conclusion: CT angiography of peripheral arteries with a low tube voltage of 70 kV and low dose of iodine provides reliable information and serves as a rapidly performed and easily available imaging modality in the diagnosis of LEASO.

First Report of Neofusicoccum parvum Causing Panicle Blight and Leaf Spot on Vitis heyneana in China.

The climbing vine, Vitis heyneana Roem. & Schult, is a member of the grape family endemic to Asia. Its fruits are used in wine production, and its roots, stems, and leaves can be used in medicinal materials. This plant is grown in Southwest China, as well as in India, Bhutan, and Nepal. Mulao Autonomous County in Guangxi Province is the only artificial cultivation area in China. During the summer of 2013, a panicle blight and leaf spot were detected on V. heyneana on four farms in Mulao Autonomous County. The symptoms were observed from the onset of florescence through fruit harvest. Brown lesions initially appeared at the base of a panicle and then extended to the whole panicle, finally causing the panicle to die and fruit to drop. When the disease developed on leaves, the symptom initially appeared as small dark brown circular spots, later enlarging into irregular spots (average diameter 6 mm) with a light brown center and dark brown rim. With severe disease, some individual leaves were affected by numerous spots, leading to premature senescence. Small sections of diseased tissue excised from 10 panicle and 10 leaf samples were plated on potato dextrose agar (PDA) and incubated at 28°C. Fungal colonies developed, initially with abundant white aerial mycelium, which turned olivaceous gray after 5 days and formed black pycnidia after 25 days. The conidia were hyaline, ellipsoidal to fusiform, externally smooth, thin-walled, and nonseptate. Thirty conidia were measured; the dimensions were 12.0 to 17.5 × 4.0 to 6.0 µm. Morphological characteristics of the isolates were similar to the descriptions of Neofusicoccum parvum (3). The isolate MPT-1 was selected as a representative for molecular identification. Genomic DNA was extracted and used for PCR to amplify the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively. The obtained ITS sequence (GenBank Accession No. KJ599627) and EF1-α sequence (KM921768) showed >99% homology with several GenBank sequences of N. parvum. Morphological and molecular results confirmed the isolate as N. parvum. For pathogenicity tests, detached, young healthy panicles and leaves of V. heyneana were surface-sterilized, wounded by sterile needle, and inoculated with mycelial plugs (3 mm in diameter) of four N. parvum isolates. Ten panicles and 10 leaves were used for every isolate. Control panicles and leaves were treated with sterile PDA plugs. All the samples were placed in a humid chamber (RH 90%, 28°C, 12 h of light) for 3 days. Symptoms similar to those observed in the field developed on all panicles and leaves inoculated with N. parvum isolates. N. parvum was reisolated from all inoculated, symptomatic tissues. The controls remained symptomless. N. parvum has been reported to cause trunk canker on V. vinifera (2), dieback on Cupressus funebris (3), and a leaf spot on Myristica fragrans (1). To our knowledge, this is the first report of N. parvum causing panicle blight and leaf spot on V. heyneana in China. Panicle blight caused a large number of fruits to drop and reduced the yield seriously. Some effective measures should be taken to control this disease. References: (1) V. Jayakumar et al. New Dis. Rep. 23:19, 2011. (2) J. Kaliternam et al. Plant Dis. 97:1656, 2013. (3) S. B. Li et al. Plant Dis. 94:641, 2010.

[Investigation and exploration of online medical education carried out by Oral and Maxillofacial Deformity Group of Shanghai Ninth People's Hospital during the COVID-19 pandemic].

As the national key discipline and the initiator of oral and maxillofacial deformity group, the Department of Oral and Maxillofacial Surgery persisted in teaching, designed a novel teaching form combining theoretical knowledge and online software practice according to the characteristics of our discipline and carried out "cloud training" via the National Oral Telemedicine Education Platform. Ten lecturers, 325 theoretical students and 50 practical students were investigated by questionnaire in the present study with questions focusing on the geographical distribution and composition of personnel, etc. The results showed that the online course covered a wide range of students and achieved high acceptance and satisfaction rate. The first online software operation course was conducted in an orderly manner, with timely interaction between teachers and students. The students were able to master the design process skillfully. This "cloud training" has achieved good results, but there are still a series of problems that have yet to be resolved, such as network stalls and protection of intellectual property rights. Under the new form, the exploration and analysis of the new mode of online telemedicine specialist education will provide some practical reference for the National Clinical Research Center for Oral Diseases to carry out online telemedicine teaching in the future.

The scid mouse as a model to identify and quantify myeloid and lymphoid stem cells.

Scid mice are excellent recipients for studying the characteristics of stem cells. Sublethal irradiation not only enhances engraftment of stem cells but enables one to graft limiting numbers of cells without compromising the survival of the recipient. This enables one to estimate the frequency of stem cells by limiting dilution analysis. Compared to fresh bone marrow, LTBMC are slightly enriched for stem cells capable of reconstituting lymphoid function in scid recipients. The stem cells have self-renewal ability since bone marrow from cured primary scid recipients can cure secondary recipients. Our results indicate that lymphoid reconstitution following engraftment with LTBMC occurs from a lymphoid-restricted stem cell; similar restricted stem cells also exist in normal bone marrow.

Hematopoietic stem cells engraft in untreated transplant recipients.

We investigated the engraftment of hematopoietic stem cells in completely untreated transplant recipients to further study hematopoietic cell regulation and for possible inclusion in gene therapy protocols. Untreated female Balb/c recipients received a single infusion of male Balb/c marrow cells. Donor origin of the hematopoietic cells was determined by polymerase chain reaction (PCR), Southern and in situ hybridization analyses with Y-chromosome-specific probes. We found that up to 47% day 12 CFU-S (26.2 +/- 12.6%, mean +/- SD, range 13.3 to 46.7%), 7.3 +/- 5.5% CFU-GM and from 2.5 to approximately 10% nucleated marrow cells were of donor-origin at 8 weeks after marrow infusion. Our results indicate that hematopoietic stem cells can stably engraft in completely unconditioned recipients but, during the interval analyzed, have a low tendency to differentiate. Moreover, the data suggest that under steady-state conditions, niches for primitive hematopoietic cells present in the marrow microenvironment are not saturated, and are readily available. We conclude that the untreated-recipient transplant model, in conjunction with sensitive techniques for the detection of donor cells, provides a valuable means for studying hematopoietic stem cell regulation and indicates a need to reassess our understanding of the interactions between stem cells and the hematopoietic microenvironment.

Hematopoietic cytokines enhance survival of SCID mice undergoing high-dose irradiation.

We have investigated the effect of hematopoietic cytokines on the survival of severe combined immune-deficient (SCID) mice that received a high dose of radiation. In this study, female SCID mice were irradiated at doses ranging from 500 to 600 cGy and then transplanted with 2 x 10(6) male Balb/c marrow cells. Groups of transplant recipients received stem cell factor (SCF), interleukin-1 (IL-1), and IL-3, alone or in combination, once daily for 5 days immediately after irradiation. Control posttransplant SCID recipients did not survive more than 2 weeks after irradiation with the dose over 500 cGy. SCF alone did not enhance survival, and treatment with IL-1 or IL-3 had very limited capacity to improve survival. IL-1 plus IL-3 has some radioprotective effect on SCID recipients, but the strongest synergistic radioprotective effect was observed in mice treated with a combination of SCF, IL-1, and IL-3. These mice survived for more than 4 months after an irradiation dose up to 600 cGy. We also examined the origin of hematopoietic stem cells in transplant recipients. Bone marrow cells were obtained from the SCID mice treated with a combination of cytokines at 2 and 4 months after transplant with male Balb/c marrow cells and irradiation with 600 cGy. These marrow cells were then transplanted into secondary lethally irradiated female Balb/c recipients. Twelve-day spleen colonies (CFU-S) were analyzed by amplification of the Y-chromosome sequence of the sex-determining region by polymerase chain reaction (PCR). All spleen colonies were of donor origin, indicating that the SCID recipients were fully reconstituted by donor cells. The results suggest that SCF, synergistic with IL-1 and IL-3, protects SCID mice from lethal doses of radiation and allows complete long-term engraftment of SCID recipients.

Synergistic effect of stem cell factor with interleukin-3 or granulocyte-macrophage colony-stimulating factor on the proliferation of murine primitive hematopoietic progenitors.

We examined the effect of recombinant murine stem cell factor (SCF) on murine primitive hematopoietic stem cells in vivo. Marrow cells from 5-fluorouracil (5-FU)-treated male CBA/J mice were transplanted into lethally irradiated female littermates. Immediately after marrow transplant, the mice received SCF, interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination daily for 6 days. Day-12 colony-forming units-spleen (CFU-S) and marrow-derived colony-forming units-granulocyte/macrophage (CFU-GM) were assessed. Bone marrow cells from primary transplant recipients were transplanted into a secondary group of lethally irradiated mice, and the number of spleen colonies arising after 12 days' engraftment was determined as pre-CFU-S. SCF alone did not increase spleen colony formation in either primary or secondary recipients. In contrast, treatment of primary recipients with SCF and GM-CSF or IL-3 or with all three cytokines resulted in a synergistic increase of CFU-S in secondary recipients, indicating increased pre-CFU-S levels. The cytokine combinations also produced synergistic increases of CFU-GM in primary recipient marrow. Evaluation of spleen colonies in secondary recipients by PCR amplification of the Y-chromosome sex-determining region indicated that about 80% were of donor (male) origin. We conclude that SCF with IL-3 and/or GM-CSF increases pre-CFU-S proliferation.

Variable self-renewal of reconstituting stem cells in long-term bone marrow cultures.

Long-term bone marrow cultures (LTBMC) serve as a valuable in vitro model of the bone marrow microenvironment. The stromal layer supports the growth of immature and mature cell populations through production of colony-stimulating factors and cell:cell interactions. LTBMC are devoid of mature lymphoid cells but contain stem cells capable of restoring lymphoid and myeloid function in suitable recipients. Severe combined immune deficiency (scid) mice provide a useful environment to study lymphocyte development, as their autosomal recessive mutation on chromosome 16 leaves them with a severe deficiency of B and T lymphocytes. To determine the presence of different classes of stem cells in LTBMC, adherent cells from the cultures were grafted into sublethally irradiated scid mice and lineage reconstitution was evaluated 6 weeks to 3 months postengraftment. Self-renewal of donor stem cells was tested by serial transfer of scid bone marrow to donor secondary C.B-17 and scid recipients. Mature lymphoid and myeloid cells were isolated from reconstituted mice and a restriction fragment length polymorphism (RFLP) at the Cmu immunoglobulin locus was used to distinguish donor and host cells. We found that LTBMC contained both long-term and short-term reconstituting stem cells. The long-term stem cells had significant self-renewal potential and fully reconstituted all lineages in both primary and secondary recipients The short-term stem cells produced mostly lymphoid progeny at the time of analysis and their limited self-renewal capacity led to partial reconstitution of only the primary recipients. The short-term reconstituting cells may be lymphoid-restricted stem cells

Skeletal effects of parathyroid hormone infusion in ovariectomized rats with or without estrogen repletion.

We employed skeletally matured rats to study changes in biochemical markers of bone turnover, bone mineral density (BMD), and bone biomechanics produced by continuous elevation of parathyroid hormone (PTH) in estrogen-deplete and -replete rodents. Ninety-six 7-month-old virgin female rats were divided randomly into 12 groups (n = 8) and treated as follows. One group was killed on the day of surgery. The remaining groups were either bilaterally ovariectomized (Ovx) or sham-operated and left untreated for 8 weeks, at which point, two groups, one sham and one Ovx, were killed. The remaining nine groups were treated for 2 weeks or 4 weeks. One sham and two Ovx groups received subcutaneous implants of Alzet miniosmotic pumps with vehicle for PTH. Two Ovx groups were given pumps with vehicle as well as a subcutaneous implant of 17beta-estradiol, which delivered 10 microg/kg per day. Two Ovx groups were implanted with rat PTH(1-34) in Alzet miniosmotic pumps, which delivered 30 microg PTH/kg per day. Two Ovx groups were implanted with both estradiol pellets and PTH-loaded pumps. One group of Ovx animals from each treatment was killed after 2 weeks and the other after 4 weeks. Biochemical markers of bone turnover, serum osteocalcin and urinary free pyridinoline, BMD, and mechanical strength of excised bones were measured. As expected, there was a significant increase in N-terminal PTH and serum calcium levels in all PTH infusion groups. Both serum osteocalcin and urinary pyridinoline showed a rapid increase within the first 2 weeks of the PTH infusion and remained elevated at week 4. In estrogen-replete groups, osteocalcin increased by week 2 of PTH infusion but pyridinoline did not increase until week 4. BMD of the distal and proximal femur showed the expected decrease 8 weeks after ovariectomy but did not exhibit any further changes during the 4 weeks of treatment with vehicle. Four weeks of PTH infusion in Ovx animals resulted in BMD loss at the midshaft, distal, and proximal regions of the femur. Estrogen repletion by itself, beginning 8 weeks after ovariectomy, produced no change in BMD at any site when compared with from Ovx vehicle-treated rats. Estrogen repletion in PTH-infused Ovx animals resulted in significant improvements of BMD comparable with sham-operated animals at all three femoral regions. The indentation test at the cancellous bone of the distal femur, three-point bending test at the midshaft femur, and cantilever bending test at the femoral neck showed that the changes in mechanical strength in these sites were consistent to the changes found in BMD. Our results showed that (1) continuously elevated levels of PTH induced additional loss of BMD in estrogen-deficient animals beyond the rapid bone loss phase associated with ovariectomy, (2) estrogen repletion, given by implant, to PTH-infused Ovx animals, reversed these BMD changes increasing BMD to levels comparable with estrogen-sufficient rats, and (3) these changes were reflected in the mechanical strength determined at these sites. These results lend experimental support that hormone replacement therapy may benefit bone health in postmenopausal women with primary hyperparathyroidism (PHPT). In addition, it raises the possibility that a continuous elevation of PTH could exert anabolic effects on skeletal tissue if its catabolic component can be minimized.

Streaming potentials in healing, remodeling, and intact cortical bone.

Electrical fields have been implicated in accelerated bone healing and as a transduction mechanism for mechanically driven bone remodeling. Applied mechanical or electrical stimulation of bone remodeling suggests that this depends on the magnitude, frequency, and duration of the stimulus. The magnitude of endogenous electrical fields, manifest by streaming potentials (SPs) across canine cortical bone, were measured as a function of bending frequency in vivo and then in vitro at healing drill holes and at remodeling (ipsilateral) and normal, intact (contralateral) control sites in canine tibia. SP magnitudes normalized to periosteal strain were smaller for drill holes at 2 and 4 weeks postsurgery relative to either remodeling (P < 0.05 at 10 Hz) or normal intact (P < 0.001 at 10 Hz) controls both in vivo and in vitro. SPs of 12 week drill holes were similar to SPs of remodeling controls and tended to be smaller than SPs of normal intact controls. Mean SP normalized to bone impedance was approximately the same for all sites, suggesting that the smaller SPs during healing and remodeling relate to smaller bone impedance and/or larger porosity. SP as a function of bending frequency for normal sites was similar to that observed previously. SP versus frequency for drill holes and remodeling controls was more variable, probably because of variations in bone microstructure, and displayed a higher frequency content. The observed differences in SP magnitude and frequency response to loading associated with stages of healing indicate that endogenous electrical fields do indeed respond to the structural changes in healing and remodeling and are therefore capable of providing structural feedback information for the repair and remodeling process.

Accretion of bone mass and strength with parathyroid hormone prior to the onset of estrogen deficiency can provide temporary beneficial effects in skeletally mature rats.

Intermittent administration of parathyroid hormone (PTH) has been shown to be an anabolic agent for animal and human skeletons. In previous studies, PTH has been used concurrent with, or subsequent to, the onset of bone loss. However, it is entirely possible that PTH may be used as an anabolic agent in a situation where there is stable skeletal remodeling. Increasing bone mass at this time might confer long-lasting beneficial effects when bone loss begins, for example, subsequent to the loss of ovarian function. To test this hypothesis, we evaluated the effects of administering rat PTH(1-34) (80 microg/kg/day, subcutaneously [s.c.]) to 6-month-old rats for a 2-week period prior to ovariectomy, and followed the natural occurrence of bone loss over a 14-week period. To determine the effects of estrogen intervention on bone gained by PTH treatment, one group was repleted with 17beta-estradiol (10 microg/kg/day via s.c. implant). Serial measurements of bone mass in vivo at the distal femur were obtained at 2-week intervals using dual-energy X-ray absorptiometry, while histologic and mechanical strength data were obtained from excised proximal tibiae and distal femurs after sacrifice. Two weeks of PTH treatment resulted in an increase of bone mineral density (BMD), mechanical strength, and cancellous bone volume (CnBV/TV). Four weeks after PTH withdrawal, significant residual beneficial effects on BMD and strength, irrespective of ovarian status, were observed. However, 14 weeks after PTH withdrawal, although there were still residual effects on CnBV/TV in ovariectomized animals pretreated with PTH, the PTH effects on BMD and mechanical strength had been lost. Estradiol repletion during the rapid bone loss phase following ovariectomy prevented the reduction in BMD associated with either ovariectomy or PTH withdrawal. Our results suggest that: treatment of rats with PTH prior to ovariectomy produces an increase in BMD and strength, these beneficial effects extend for a period of at least three times the treatment duration, the BMD that is lost when PTH is discontinued equates to the amount accrued during the PTH treatment, estrogen replacement can be used to maintain the bone gained as a result of PTH treatment.

Temporal expression of the anabolic action of PTH in cancellous bone of ovariectomized rats.

When administered intermittently, parathyroid hormone (PTH) is a potent anabolic agent in both human and animal bone. To improve our understanding of this anabolic effect, we have examined the time course of PTH action in an established animal model of estrogen deficiency-induced bone loss: the ovariectomized rat. Animals were ovariectomized (Ovx) and allowed to lose bone for 6 weeks. A dose of 20 micrograms/kg/d of rat PTH (1-34) was administered s.c., 6 days each week for periods of 1, 2, 3, 4, 6 and 8 weeks. Animals were sacrificed for evaluation of skeletal histomorphometry of the proximal tibia and mechanical strength of the cancellous bone in the marrow cavity of the distal femur. Cancellous bone volume (Cn-BV/TV) increased gradually over 8 weeks of treatment (16.8 +/- 1.6 to 24.1 +/- 2.7%) as did the bone formation rate (0.308 +/- 0.054 to 1.659 +/- 0.293 microns3/micron2/d), as determined by an increase in both total mineralization surface (15.5 +/- 2.1 to 42.7 +/- 5.0%) and mineral apposition rate (1.88 +/- 0.20 to 3.55 +/- 0.39 microns/d). The largest increments in these variables reflecting bone formation occurred over the first week of treatment. This bone formation was accompanied by an increase in trabecular thickness (Tb.Th) (55.3 +/- 3.4 to 80.5 +/- 5.0 microns) without a corresponding increment in trabecular number (Tb.N) (3.65 +/- 0.17 to 3.55 +/- 0.26). Extensive tetracycline labels were visualized on the surface of trabecular rod-like and plate-like structures. A small transient, though not statistically significant, increase occurred in both eroded surface and urinary pyridinoline concentration immediately after the onset of PTH administration. Osteocalcin showed a small decrement in the first two weeks after PTH administration, but the levels were elevated when compared with the Ovx control in later weeks. Mechanical strength of the cancellous bone also increased significantly with PTH treatment (20.5 +/- 2.4 to 46.1 +/- 10.0 Newtons). Our results showed that: 1) intermittent PTH treatment of Ovx rats elicited an immediate increase of bone formation activity by the existing osteoblasts, 2) the increase of Cn-BV/TV after PTH administration resulted primarily from an increase in Tb.Th, and 3) improved mechanical strength after PTH treatment can be achieved by increases in Tb.Th without an increase in Tb.N.

Prednisolone alone, or in combination with estrogen or dietary calcium deficiency or immobilization, inhibits bone formation but does not induce bone loss in mature rats.

Glucocorticoid use has long been recognized as a risk factor for bone loss, resulting in an increased fracture incidence in humans. However, steroid-treated patients often present with other complications that predispose to bone loss, such as immobilization, and little is known about the interaction of these other risk factors for bone loss and glucocorticoids. In the present study, mature female rats were treated with prednisolone (Pred) or vehicle, in combination with ovariectomy (ovx), dietary calcium deficiency (LoCa), or right hind limb immobilization (IM). After 4 weeks of treatment, the rats were killed and the right tibia and tibiofibular junction were collected for quantitative histomorphometric analysis and the right femur was collected for bone mineral density (BMD) and mechanical strength determinations. As expected, ovx, LoCa, and IM decreased BMD in the distal femur and cancellous bone volume (CnBV/TV) in the proximal tibia. All Pred-treated groups responded with increases of BMD and CnBV/TV, when compared to their respective non-Pred treated groups. Mechanical strength testing of the cancellous bone of the distal femur reflected the changes in BMD and CnBV/TV. No differences in trabecular plate thickness were noted in any of the treatment groups. The Pred group showed a significant reduction in longitudinal growth rate, as well as bone formation rate (BFR/BS), in the proximal tibia when compared with their respective control groups, the latter indicated by a decrease in both mineralizing surface and mineral apposition rate. Most notably, osteoclast surface and urinary free pyridinoline, a bone resorption marker, increased significantly with each of the three risk factors. Pred treatment inhibited these increases but it did not exert significant reductions when used by itself. At the tibiofibular junction, there were no measurable changes in either total bone or cortical bone area. Endocortical BFR/BS were increased by ovx or LoCa but each was lowered by Pred treatment. Periosteal BFR/BS were increased by ovx and IM, and Pred exerted significant inhibition by itself and in combination with other risk factors. We conclude, therefore, that unlike the effects observed in humans treated with glucocorticoid, treatment of rats with prednisolone not only does not result in bone loss but may exert a protective effect on the skeleton through the inhibition of bone resorption.

The effects of altered strain environments on bone tissue kinetics.

This study defines the alteration in bone tissue kinetics responsible for the "adaptive remodeling" response to altered strain environments. Adult beagle dogs were separated into three experimental groups: ulnar osteotomy, ulnar osteotomy with fracture fixation plate spanning the gap and sham surgery. Four sets of double fluorochrome labels were administered. Prior to sacrifice at 1, 3, and 6 months, strains were measured through rosette strain gages on the cranial and caudal surfaces of the intact radius. Histomorphometric analysis indicated that the increased bone mass in response to elevated strain results from increased activation frequency of modeling with more sites undergoing formation processes than resorption processes on periosteal and endocortical surfaces. Increased remodeling activation did not lead to increased bone mass. There was no evidence that elevated strain changes the individual vigor of osteoclasts or osteoblasts, or that the sigma period was altered by elevated strain.

Short-term immobilization-induced cancellous bone loss is limited to regions undergoing high turnover and/or modeling in mature rats.

Estrogen and calcium deficiencies increase both bone resorption and formation, whereas immobilization mainly decreases bone formation. How these functionally different risk factors for bone loss interact in cancellous bone undergoing modeling or remodeling activity is not well understood. Mature (6-month-old) female rats were subjected to sham operation (sham), ovariectomy (ovx), dietary calcium deficiency (LoCa, 0.1% Ca), and sciatic and femoral denervation (IM), ovx+IM, or LoCa+IM for 4 weeks. The primary spongiosa, the region of active modeling within 1 mm of the growth plate, in ovx, LoCa, and IM groups showed a decrease in cancellous bone volume, trabecular number, and connectivity when compared to sham controls. Groups combining two risk factors exhibited additive changes when compared with single risk factor groups. In the secondary spongiosa, an area with little modeling activity, ovx and LoCa groups, as expected, lost bone. In contrast with the primary spongiosa, IM alone did not induce bone loss in the secondary spongiosa, and the groups with a combination of IM and ovx or IM and LoCa showed a greater bone loss than either ovx or LoCa alone. Ovx and LoCa groups showed increases in both bone formation rate and eroded surface in the secondary spongiosa, while IM groups showed a decrease in bone formation rate. Combining IM with either ovx or LoCa resulted in increased eroded surface. The effects on cortical bone were assessed at the tibio-fibular junction. A trend toward decreased percentage of cortical bone area and an increase in marrow cavity area were observed in the combined deficiency groups only. These changes were the result of a statistically significant increase in endosteal eroded surface in IM+ovx and IM+LoCa groups. Our results demonstrate that immobilization-induced bone loss is restricted to the primary spongiosa where most modeling events occur. However, the inhibitory effect of IM on bone formation in the secondary spongiosa is unmasked in remodeling sites when a high turnover state is provided by either estrogen or dietary calcium deficiency. These results suggest that the presence of a risk factor, such as immobilization, which in the short-term causes inhibition of bone formation, does not predispose the skeleton to rapid cancellous bone loss except when accompanied by modeling or high turnover.