Beneficial Effects of Tocotrienol and Tocopherol on Bone Histomorphometric Parameters in Sprague–Dawley Male Rats After Nicotine Cessation

This study was conducted to determine the effectiveness of three forms of vitamin E supplements following nicotine treatment on bone histomorphometric parameters in an adult male rat model. Rats were divided into seven groups: baseline (B, killed without treatment), control (C, normal saline for 4 months), nicotine (N, nicotine for 2 months), nicotine cessation (NC), tocotrienol-enhanced fraction (TEF), gamma-tocotrienol (GTT), and alpha-tocopherol (ATF). Treatments for the NC, TEF, GTT, and ATF groups were performed in two phases. For the first 2 months they were given nicotine (7 mg/kg), and for the following 2 months nicotine administration was stopped and treatments with respective vitamin E preparations (60 mg/kg) were commenced except for the NC group, which was allowed to recover without treatment. Rats in the N and NC groups had lower trabecular bone volume, mineral appositional rate (MAR), and bone formation rate (BFR/BS) and higher single labeled surface and osteoclast surface compared to the C group. Vitamin E treatment reversed these nicotine effects. Both the TEF and GTT groups, but not the ATF group, had a significantly higher trabecular thickness but lower eroded surface (ES/BS) than the C group. The tocotrienol-treated groups had lower ES/BS than the ATF group. The GTT group showed a significantly higher MAR and BFR/BS than the TEF and ATF groups. In conclusion, nicotine induced significant bone loss, while vitamin E supplements not only reversed the effects but also stimulated bone formation significantly above baseline values. Tocotrienol was shown to be slightly superior compared to tocopherol. Thus, vitamin E, especially GTT, may have therapeutic potential to repair bone damage caused by chronic smoking.     

Specific Effects of γ-Linolenic,Eicosapentaenoic, and Docosahexaenoic Ethyl Esters on Bone Post-ovariectomy in Rats

Long chain polyunsaturated fatty acids (LCPUFAs) are involved in the regulation of bone metabolism. Increased dietary consumption of n-3, and possibly some n-6, LCPUFAs may limit postmenopausal bone loss. The aim of this study was to determine the effects on bone of specific fatty acids within the n-3 and n-6 LCPUFA families in ovariectomized (OVX) rats. Rats were OVX or sham-operated and fed either a control diet (OVX and sham) or a diet supplemented with 0.5 g/kg body weight/day of γ-linolenic (GLA), eicosapentaenoic (EPA), docosahexaenoic (DHA) ethyl esters or a mixture of all three (MIX) for 16 weeks. Bone mineral content (BMC), area, and density and plasma concentrations of insulin-like growth factor-I, vitamin D, selected biochemical markers of bone metabolism, and parathyroid hormone (PTH) were determined. The OVX-induced decrease in lumbar spine BMC was significantly attenuated by DHA but not by EPA or GLA supplementation or supplementation with a mixture of all three LCPUFAs. Endosteal circumferences of tibiae were significantly greater in DHA and EPA compared to OVX. Plasma C-terminal telopeptide of type I collagen and osteocalcin concentrations were not significantly different in the DHA group compared to OVX. Femur BMC decreased by a significantly greater amount in GLA than OVX, and final plasma PTH concentrations were significantly higher in GLA compared to all other groups. In conclusion, DHA ameliorated OVX-induced bone mineral loss. GLA exacerbated post-OVX bone mineral loss, possibly as a result of PTH-induced bone catabolism.     

Is Nitric Oxide a Mediator of the Effects of Low-Intensity Electrical Stimulation on Bone in Ovariectomized Rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvão et al., Calcif Tissue Int 84:502–509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by l-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200–220 g) were divided into six groups: sham, sham-l-NAME (6 mg/kg/day), OVX, OVX-l-NAME, OVX-LIES, and OVX-LIES-l-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of l-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). l-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both l-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats l-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.     

Histology surrounding cystotomy healing in a Sprague–Dawley rat model

Introduction and hypothesis

The purpose of this study was to histologically chronicle wound healing following cystotomy repair using a small animal model.

Methods

Thirty female Sprague–Dawley rats were included in this study. Twenty-eight rats underwent a vertical cystotomy in the bladder dome, which was repaired in a single continuous fashion. Two rats served as histological controls. Following cystotomy repair, groups of three to four rats were studied at single day intervals for 4 days, then at 2-day intervals until 10 days post-repair. The animal bladders were harvested and examined for inflammation, scar formation, and bladder healing.

Results

Thirty rat bladders were histologically examined. An inflammatory wound phase was observed during the first 4 days after wounding. Transition from acute to chronic inflammation was observed at day 2 with chronic inflammation persisting through day 10. Inflammation severity peaked 4 days post-wounding without regression through day 10. Evidence of proliferative phase wound healing was first observed 4 days post-wounding.

Conclusion

Early increases in wound healing are due to inflammatory events such as fibrin plugging of the wound. Later developments after day 4 are due to wound proliferation, collagen deposition, and re-epithelialization. Additionally, wound healing in the rat bladder is observed on a continuum and not necessarily in discrete stages observed on precisely the same postoperative day in each animal.     

Consumption of krill protein concentrate prevents early renal injury and nephrocalcinosis in female Sprague–Dawley rats

Female Sprague–Dawley rats provide an animal model for studying the role of nutrition in renal health due to their sensitivity to diet-induced alterations in kidney function. Nephrocalcinosis, a common renal abnormality found in rats, has been implicated in subsequent renal failure. Simple dietary manipulations, such as changing the source of dietary protein, may influence nephrocalcinosis. This study evaluates the consumption of krill protein concentrate (KPC), a novel and high-quality protein, on renal and bone health. Young female Sprague–Dawley rats (n = 10/group) were individually housed in metabolic cages and fed ad libitum diets consisting of 10% crude protein supplied as KPC or casein for 4 weeks. Diets were isocaloric, isonitrogenous, and matched for calcium (Ca) and phosphorus (P). Urinary n-acetyl glucosaminidase (NAG) was measured and kidney histology performed to assess kidney damage. Biomarkers of kidney function were determined by calorimetric assays. Ca and P balance and bone concentrations were measured using inductively coupled plasma mass spectrometry. Femoral strength was determined by three-point bend testing. Rats fed KPC had lower (P = 0.005) urinary NAG levels and minimal microtubular Ca deposition compared to rats fed casein. There was a tendency (P < 0.06) for higher glomerular filtration rates and lower proteinuria, and higher (P = 0.03) urinary output in rats fed KPC compared to casein. There were no differences in Ca and P balance or bone measurements of total bone mineral content, Ca, P or strength between rats fed KPC and casein. Based on the study results, KPC prevented early renal injury leading to nephrocalcinosis and potential bone loss.     

Radioprotectant and Radiosensitizer Effects on Sterility of γ-irradiated Bone

Gamma radiation is widely used to sterilize bone allografts but may impair their strength. While radioprotectant use may reduce radiation damage they may compromise sterility by protecting pathogens. We assessed the radioprotective potential of various agents (L-cysteine, N-acetyl-L-cysteine, L-cysteine-ethyl-ester and L-cysteine-methyl-ester) to identify those which do not protect spores of Bacillus subtilis. We hypothesized charge of these agents will affect their ability to radioprotect spores. We also determined ability of these radioprotectants and a radiosensitizer (nitroimidazole-linked phenanthridinium) to selectively sensitize spores to radiation damage by intercalating into the nucleic acid of spores. Spores were treated either directly in solutions of these agents or treated after being embedded and sealed in bone to assess the ability of these agents to diffuse into bone. L-cysteine and L-cysteine-ethyl-ester did not provide radioprotection. Positively charged L-cysteine-methyl-ester protected the spores, whereas positively charged L-cysteine-ethyl-ester did not, indicating charge does not determine the extent of radioprotection. The spores were sensitized to radiation damage when irradiated in nitroimidazole-linked phenanthridinium solution and sensitization disappeared after rinsing, suggesting nitroimidazole-linked phenanthridinium was unable to intercalate into the nucleic acid of the spores. Some cysteine-derived radioprotectants do not shield bacterial spores against gamma radiation and may be suitable for curbing the radiation damage to bone grafts while achieving sterility. One or more of the authors (OA, JS, SAK) has received funding from a research grant from the Musculoskeletal Transplant Foundation.     

Effects of 830 and 670 nm Laser on Viability of Random Skin Flap in Rats

Abstract Objective: This study aimed to investigate the effect of 830 and 670?nm diode laser on the viability of random skin flaps in rats. Background data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and reducing the inflammatory process after injury. However, the efficiency of such treatment remains uncertain, and there is also some controversy regarding the efficacy of different wavelengths currently on the market. Materials and methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group, group 2 received 830?nm laser radiations, and group 3 was submitted to 670?nm laser radiation (power density=0.5 mW/cm(2)). The animals underwent laser therapy with 36?J/cm(2) energy density (total energy=2.52?J and 72?sec per session) immediately after surgery and on the 4 subsequent days. The application site of laser radiation was one point at 2.5?cm from the flap's cranial base. The percentage of skin flap necrosis area was calculated on the 7th postoperative day using the paper template method. A skin sample was collected immediately after to determine the vascular endothelial growth factor (VEGF) expression and the epidermal cell proliferation index (KiD67). Results: Statistically significant differences were found among the percentages of necrosis, with higher values observed in group 1 compared with groups 2 and 3. No statistically significant differences were found among these groups using the paper template method. Group 3 presented the highest mean number of blood vessels expressing VEGF and of cells in the proliferative phase when compared with groups 1 and 2. Conclusions: LLLT was effective in increasing random skin flap viability in rats. The 670?nm laser presented more satisfactory results than the 830?nm laser.     

Effects of Aloe Vera on Spinal Cord Ischemia–Reperfusion Injury of Rats

Aim: The purpose of this study was to evaluate the possible protective/therapeutic effects of aloe vera (AV) on ischemia–reperfusion injury (I/R) of spinal cord in rats. Materials and Methods: A total of 28 Wistar Albino rats were divided into four random groups of equal number (n = 7). Group I (control) had no medication or surgery; Group II underwent spinal cord ischemia and was given no medication; Group III was administered AV by gastric gavage for 30 days as pre-treatment; Group IV was administered single dose intraperitoneal methylprednisolone (MP) after the ischemia. Nuclear respiratory factor-1 (NRF1), malondialdehyde (MDA) and superoxide dismutase (SOD) levels were evaluated. Tissue samples were examined histopathologically and neuronal nitric oxide synthase (nNOS) and nuclear factor-kappa B (NF-κB) protein expressions were assessed by immunohistochemical staining. Results: NRF1 and SOD levels of ischemia group were found to be lower compared to the other groups. MDA levels significantly increased after I/R. Treatment with AV and MP resulted in reduced MDA levels and also alleviated hemorrhage, edema, inflammatory cell migration and neurons were partially protected from ischemic injury. When AV treatment was compared with MP, there was no statistical difference between them in terms of reduction of neuronal damage. I/R injury increased NF-κB and nNOS expressions. AV and MP treatments decreased NF-κB and nNOS expressions.Conclusions: It was observed that aloe vera attenuated neuronal damage histopathologically and biochemically as pretreatment. Further studies may provide more evidence to determine the additional role of aloe vera in spinal cord ischemia reperfusion injury.     

Does Exercise Modify the Effects of Zoledronic Acid on Bone Mass,Microarchitecture, Biomechanics,and Turnover in Ovariectomized Rats?

Regular activity has effects on bone size, shape, and density, resulting in an increase in mechanical strength. The mechanism of action that underlies this improvement in bone strength is mainly linked to an increase in bone formation. Zoledronic acid (Z), in contrast, may prevent bone strength changes in ovariectomized (OVX) rodents by its potent antiresorptive effects. Based on these assumptions we hypothesized that combined effects of exercise (E) and Z may produce higher benefits on bone changes resulting from estrogen deficiency than either intervention alone. At 6 months of age, 60 female Wistar rats were OVX or sham operated (SH) and divided into five groups: SH, OVX, OVX-E, OVX-Z, and OVX-ZE. OVX rats were treated with a single IV injection of Z (20 μg/kg) or vehicle and submitted or not to treadmill exercise (15 m/min, 60 min/day, 5 days/week) for 12 weeks. Whole-body BMD and bone turnover markers were analyzed longitudinally. At sacrifice, femurs were removed. BMD by DXA, three-point bending test, and μCT were performed to study biomechanical and trabecular structure parameters, respectively. After 12 weeks, bone volume fraction decreased in OVX rats, whereas bone turnover rate, trabecular spacing, and structure model index increased compared with those in the SH group (P < 0.05). Zoledronic acid prevented the ovariectomy-induced trabecular bone loss and its subsequent trabecular microarchitectural deterioration. Treadmill exercise running was shown to preserve the bone strength and to induce bone turnover changes in favor of bone formation. However, the combined effects of zoledronic acid and running exercise applied simultaneously did not produce any synergetic or additive effects.     

Effects of Transfusion of Stored Red Blood Cells on Renal Ischemia-Reperfusion–Induced Hepatic Injury in Rats

BackgroundRenal ischemia-reperfusion (IR) injures the liver as well as the kidneys. Transfusion of stored red blood cells (RBCs) triggers inflammatory responses, oxidative stress, and activation of innate immunity. In the present study, we investigated the effect of transfusion of stored RBCs on renal IR-induced hepatic injury.MethodsSprague–Dawley rats were randomly divided into 3 groups based on the following treatments: rats subjected to sham operation (sham group), rats subjected to the induction of renal IR only (RIR group), and rats transfused with stored RBCs 1 hour after the start of reperfusion (RIR-TF group). Renal ischemia was induced for 1 hour, and reperfusion was allowed for 24 hours. After reperfusion, blood and liver tissue samples were obtained.ResultsSerum levels of aspartate and alanine aminotransferase were increased in the RIR-TF group compared with those in the RIR and sham groups. The hepatic mRNA expression levels of heme oxygenase-1 and neutrophil gelatinase-associated lipocalin were increased in the RIR-TF group compared with those in the RIR and sham groups. The mRNA expression level of high mobility group box-1 was also increased in the RIR-TF group compared with that in the RIR group.ConclusionThe transfusion of stored RBCs exacerbates renal IR-induced liver damage. Oxidative stress may be responsible for hepatic injury.     

Impact of Smoking and Smoking Cessation on Oncologic Outcomes in Primary Non–muscle-invasive Bladder Cancer

Background

Cigarette smoking is the best-established risk factor for urothelial carcinoma (UC) development, but the impact on oncologic outcomes remains poorly understood.

Objective

To analyse the effects of smoking status, cumulative exposure, and time from smoking cessation on the prognosis of patients with primary non–muscle-invasive bladder cancer (NMIBC).

Design, setting, and participants

We collected smoking data from 2043 patients with primary NMIBC. Smoking variables included smoking status, average number of cigarettes smoked per day (CPD), duration in years, and time since smoking cessation. Lifetime cumulative smoking exposure was categorised as light short term (≤19 CPD, ≤19.9 yr), light long term (≤19 CPD, ≥20 yr), heavy short term (≥20 CPD, ≤19.9 yr) and heavy long term (≥20 CPD, ≥20 yr). The median follow-up in this retrospective study was 49 mo.

Interventions

Transurethral resection of the bladder with or without intravesical instillation therapy.

Outcome measurements and statistical analysis

Univariable and multivariable logistic regression and competing risk regression analyses assessed the effects of smoking on outcomes.

Results and limitations

There was no difference in clinicopathologic factors among never (24%), former (47%), and current smokers (29%). Smoking status was associated with the cumulative incidence of disease progression in multivariable analysis (p = 0.003); current smokers had the highest cumulative incidences. Among current and former smokers, cumulative smoking exposure was associated with disease recurrence (p < 0.001), progression (p < 0.001), and overall survival (p < 0.001) in multivariable analyses that adjusted for the effects of standard clinicopathologic factors and smoking status; heavy long-term smokers had the worst outcomes, followed by light long-term, heavy short-term, and light short-term smokers. Smoking cessation >10 yr reduced the risk of disease recurrence (hazard ratio [HR]: 0.66; 95% confidence interval [CI], 0.52–0.84; p < 0.001) and progression (HR: 0.42; 95% CI, 0.22–0.83; p = 0.036) in multivariable analyses. The study is limited by its retrospective nature.

Conclusions

Smoking status and a higher cumulative smoking exposure are associated with worse prognosis in patients with NMIBC. Smoking cessation >10 yr abrogates this detrimental effect. These findings underscore the need for integrated smoking cessation and prevention programmes in the management of NMIBC patients.     

Histomorphometric Evaluation of the Recovering Effect of Human Parathyroid Hormone (1–34) on Bone Structure and Turnover in Streptozotocin-Induced Diabetic Rats

The purpose of this study was to determine whether human parathyroid hormone (h-PTH) enhances trabecular bone mass and connectivities that were reduced by streptozotocin (STZ)-induced diabetes in rats. Seven-month-old female Wistar rats were injected with STZ or its vehicle intraperitoneally. All vehicle-injected normal controls were sacrificed 0, 4, 6, 8, 12, 14, and 16 weeks after injection, and one-third of the STZ-injected rats were sacrificed as the baseline controls 4, 6, and 8 weeks after the injection. Eight-week h-PTH (6.0 μg/kg, 6 times a week) or its vehicle treatment by subcutaneous injection for residual diabetic rats was started 4, 6, or 8 weeks after the STZ injection. The rats' proximal right tibiae were processed for undecalcified Villanueva bone staining sections for bone histomorphometry. Furthermore, changes in trabecular connectivities were determined by node-strut analysis. The decreased cancellous bone volume (BV/TV) and turnover in diabetic rats were recovered in all PTH-treated groups. In node-strut analysis, the node-related parameters (N.Nd/TV, NdNd/TV) were significantly increased by PTH when it was administered 4 weeks after STZ injection but were not increased when administration was started after 6 weeks. The results indicated that PTH enhanced bone turnover and bone mass but not trabecular connectivity in the late stage of diabetes in rats. Early treatment of osteoporosis is important in preventing fractures caused by decreased bone strength resulting from decreased trabecular connectivity. Received: 11 May 1999 / Accepted: 20 September 1999     

Effects of Treatment with Parathyroid Hormone 1–84 on Quantity and Biomechanical Properties of Thoracic Vertebral Trabecular Bone in Ovariectomized Rhesus Monkeys

Osteoporosis is characterized by impaired bone quality leading to increased susceptibility to fracture, particularly of the thoracic spine. However, the lumbar spine is studied most commonly. We investigated the effects of 16 months of treatment with full-length parathyroid hormone (PTH) 1–84 (5, 10, or 25 μg/kg) on bone mineral density (BMD) and on architecture and biomechanical properties of trabecular bone at the thoracic spine of ovariectomized (OVX) adult rhesus monkeys and compared the results with those from the lumbar spine. At baseline, 9 months after surgery, dual-energy X-ray absorptiometric BMD at T9–T12 was 7% lower in OVX than in sham animals. All PTH(1–84) doses increased BMD to sham levels within 7 months. Micro-computed tomography of T10 vertebrae showed that trabecular bone volume and connectivity were higher in PTH(1–84)-treated animals than in sham controls, primarily through a significantly greater trabecular number. Peripheral quantitative computed tomography of trabecular bone cores from T11 and T12 confirmed that PTH(1–84) increased BMD. Compression testing of the cores showed that PTH(1–84) treatment increased stiffness, modulus, yield load, and yield stress to levels significantly greater than in sham animals, with the largest effect in the 10 μg/kg group (35–54% greater than in OVX controls). Thus, PTH(1–84) treatment increased BMD and the biomechanical properties of trabecular bone at the thoracic spine of OVX rhesus monkeys. The 10 μg/kg dose produced the greatest effect on trabecular strength, possibly because the highest dose stimulated bone remodeling excessively. Importantly, the changes observed were similar to those in lumbar vertebrae, thereby validating extrapolation of results from the lumbar to the thoracic spine.     

Treatment of Human Immunodeficiency Virus Infection With Tenofovir Disoproxil Fumarate–Containing Antiretrovirals Maintains Low Bone Formation Rate,But Increases Osteoid Volume on Bone Histomorphometry

Bone mineral density (BMD) loss is a known complication of human immunodeficiency virus (HIV) infection and its treatment, particularly with tenofovir disoproxil fumarate (TDF)-containing antiretroviral regimens. Although renal proximal tubular dysfunction and phosphaturia is common with TDF, it is unknown whether BMD loss results from inadequate mineralization. We evaluated change in BMD by dual-energy X-ray absorptiometry (DXA) and bone histomorphometry by tetracycline double-labeled transiliac crest biopsies in young men living with HIV before (n = 20) and 12 months after (n = 16) initiating TDF/lamivudine/efavirenz. We examined relationships between calciotropic hormones, urinary phosphate excretion, pro-inflammatory and pro-resorptive cytokines, and bone remodeling-related proteins with changes in BMD and histomorphometry. Mean age was 29.6 ± 5.5 years, with mean CD4 + T cell count of 473 ± 196 cells/mm³. At baseline, decreased bone formation rate and increased mineralization lag time were identified in 16 (80%) and 12 (60%) patients, respectively. After 12 months, we detected a 2% to 3% decrease in lumbar spine and hip BMD by DXA. By histomorphometry, we observed no change in bone volume/total volume (BV/TV) and trabecular parameters, but rather, increases in cortical thickness, osteoid volume, and osteoblast and osteoclast surfaces. We did not observe significant worsening of renal phosphate excretion or mineralization parameters. Increases in PTH correlated with decreased BMD but not histomorphometric parameters. Overall, these data suggest abnormalities in bone formation and mineralization occur with HIV infection and are evident at early stages. With TDF-containing antiretroviral therapy (ART), there is an increase in bone remodeling, reflected by increased osteoblast and osteoclast surfaces, but a persistence in mineralization defect, resulting in increased osteoid volume. © 2019 American Society for Bone and Mineral Research.     

Screening Gene Knockout Mice for Variation in Bone Mass: Analysis by μCT and Histomorphometry

Purpose of review

The international mouse phenotyping consortium (IMPC) is producing defined gene knockout mouse lines. Here, a phenotyping program is presented that is based on micro-computed tomography (μCT) assessment of distal femur and vertebra. Lines with significant variation undergo a computer-based bone histomorphometric analysis.

Recent findings

Of the 220 lines examined to date, approximately 15% have a significant variation (high or low) by μCT, most of which are not identified by the IMPC screen. Significant dimorphism between the sexes and bone compartments adds to the complexity of the skeletal findings. The μCT information that is posted at www.bonebase.org can group KOMP lines with similar morphological features. The histological data is presented in a graphic form that associates the cellular features with a specific anatomic group.

Summary

The web portal presents a bone-centric view appropriate for the skeletal biologist/clinician to organize and understand the large number of genes that can influence skeletal health. Cataloging the relative severity of each variant is the first step towards compiling the dataset necessary to appreciate the full polygenic basis of degenerative bone disease.