An external fixation method and device to study fracture healing in rats

We wished to establish a reproducible model for fracture fixation to be used in fracture healing research and therefore developed an external fixation construct and surgical procedure adapted to Sprague-Dawley rats. We evaluated the mechanical properties of the construct in brass rods and rat bone, in an Instron test machine with axial and transverse loading, and the in vivo performance. We found that the mechanical properties of the construct in brass rods were predictable and could be repeated in rat femora. In all tests, the axial load was about 10 times the transverse for the same degree of deformation. The stiffness among fixators was uniform. 1 mm pins caused about 50% less stiffness than 1.2 mm pins in axial loading of rat bone (p < 0.001) and brass rods (p < 0.001) as well as in transverse loading of brass rods (p < 0.001). Loosening of 1 or 2 screws that lock the pins to the fixator reduced stiffness by about 50% in axial loading of rat bone (p = 0.009) and brass rods (p = 0.05). A change in the distance between the bone surface and the fixator was linearly related to the stiffness in axial loading of rat bone (p < 0.001) and brass rods (p < 0.001) and in transverse loading of brass rods (p < 0.001). If the bone ends touched each other, the axial stiffness of the construct increased almost 10 times (265 N/mm), as compared to a fracture gap size of 2 mm (31 N/mm). In vivo experiments had a complication rate of less than 10% when we used 1.2 mm pins, 6 mm offset and rats weighing 350-450 g. Our method and device for experimental external fixation of rat femora are reliable and the findings are reproducible. These can be used in bone repair and fracture healing research.     

The AGE inhibitor pyridoxamine inhibits lipemia and development of renal and vascular disease in Zucker obese rats

BACKGROUND: In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. METHODS: Three groups of Zucker rats were studied: lean (Fa/fa), untreated fatty (fa/fa), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. RESULTS: AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. CONCLUSION: Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.     

Improved Scoring System to Assess Adult Donors For Cadaver Renal Transplantation

We previously proposed a quantitative approach to assess donor organs for cadaver renal transplantation. To improve on our original scoring system, we studied 34 324 patients who received cadaver renal transplants from adult donors between 1994 and 1999 and were reported to the UNOS Scientific Renal Transplant Registry. A scoring system was developed from five donor variables (age, 0-25 points; history of hypertension, 0-4; creatinine clearance before procurement, 0-4; cause of death, 0-3; HLA mismatch, 0-3) that showed a significant correlation with renal function and long-term graft survival. Cadaver kidneys were stratified by cumulative donor score: grade A, 0-9 points; grade B, 10-19; grade C, 20-29; and grade D, 30-39. The influence of donor score on renal function and graft survival was most severe above 20 points, designated 'marginal' kidneys. In summary, a donor scoring system developed from a large population database was useful in predicting outcome after cadaver renal transplantation. The improved system provides a quantitative approach to evaluation of marginal kidneys and may improve allocation of these organs in cadaver renal transplantation.     

Adjuvant hepatic arterial chemotherapy following metastasectomy in patients with isolated liver metastases

OBJECTIVE: To examine survival and toxicity by querying a single-institutional experience with adjuvant hepatic arterial infusional (HAI) chemotherapy. SUMMARY BACKGROUND DATA: Three randomized series in the literature have examined adjuvant HAI after complete resection of liver metastases. Only one of these trials showed an overall survival benefit at 2 years but not over the entire time period of the study. Previous studies in patients with unresectable disease were plagued by high rates of biliary toxicity. METHODS: A retrospective review of a prospectively maintained database was performed. Hepatic arterial pumps were placed in the standard fashion. Patients received floxuridine at doses previously demonstrated as safe in the literature. Standard statistical methods were used. RESULTS: Twenty-one of 92 patients underwent placement of hepatic arterial pumps at the time of liver resection. The HAI group was similar in all demographic measures to the non-HAI group, with the exception that the HAI patients were significantly younger. No differences were seen between the groups in either disease-free or overall survival, although a trend toward improved hepatic progression-free survival was noted. Significant biliary sclerosis developed in six patients in the HAI group, requiring chronic indwelling stents in four patients. One patient died of progressive liver failure associated with this toxicity. CONCLUSIONS: Biliary toxicity is an important potential side effect of hepatic arterial chemotherapy. Although larger randomized studies and this one suggest significant improvements in hepatic recurrences, these have not reliably translated into overall survival benefit. This fact, in light of the potential toxicity, would argue for a larger confirmatory trial of HAI in the adjuvant setting, incorporating recent advances in systemic therapy and careful attention to hepatotoxicity.     

Symptom burden, quality of life, advance care planning and the potential value of palliative care in severely ill haemodialysis patients.

BACKGROUND: There has been little research on the potential value of palliative care for dialysis patients. In this pilot study, we sought (i) to identify symptom burden, health-related quality of life (HRQoL) and advance directives in extremely ill haemodialysis patients to determine their suitability for palliative care and (ii) to determine the acceptability of palliative care to patients and nephrologists. METHODS: Nineteen haemodialysis patients with modified Charlson co-morbidity scores of > or =8 were recruited. Each completed surveys to assess symptom burden, HRQoL and prior advance care planning. Palliative care specialists then visited patients twice and generated recommendations. Patients again completed the surveys, and dialysis charts were reviewed to assess nephrologists' (i) compliance with recommendations and (ii) documentation of symptoms reported by patients on the symptom assessment survey. Patients and nephrologists then completed surveys assessing their satisfaction with palliative care. RESULTS: Patients reported 10.5 symptoms, 40% of which were noted by nephrologists in patients' charts. HRQoL was significantly impaired. Thirty-two percent of patients had living wills. No differences were observed in symptoms, HRQoL or number of patients establishing advance directives as a result of the intervention. Sixty-eight percent of patients and 76% of nephrologists rated the intervention worthwhile. CONCLUSIONS: Extremely ill dialysis patients have marked symptom burden, considerably impaired HRQoL and frequently lack advance directives, making them appropriate candidates for palliative care. Patients and nephrologists perceive palliative care favourably despite its lack of effect in this study. A more sustained palliative care intervention with a larger sample size should be attempted to determine its effect on the care of this population.     

Two-dimensional gel electrophoresis of synovial fluid: method for detecting candidate protein markers for osteoarthritis

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and -globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89–0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85–0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.     

Selective use of esophageal manometry and 24-Hour pH monitoring before laparoscopic fundoplication

BACKGROUND: Preoperative esophageal manometry and 24-hour pH monitoring commonly are used in preoperative evaluation of patients undergoing fundoplication. Here we review our experience with the selective preoperative workup of patients undergoing fundoplication to treat gastroesophageal reflux disease. STUDY DESIGN: A series of 628 consecutive antireflux procedures was reviewed. History and physical examination, upper endoscopy, and upper gastrointestinal videofluoroscopy were obtained preoperatively on all patients; the first 30 patients also underwent esophageal manometry and pH monitoring (routine evaluation group). Thereafter, pH monitoring only was performed for atypical reflux symptoms, and manometry only was performed for a history of dysphagia, odynophagia, or for abnormal motility on videofluoroscopy (selective evaluation group). All patients underwent a laparoscopic floppy Nissen fundoplication, and then endoscopy and fluoroscopy at 3 months and 12 months postoperatively. RESULTS: Eighty-five of the patients in the selective evaluation group (14%) required manometry, and 88 (15%) underwent pH monitoring. Eighteen of the 115 patients who underwent manometry (16%) had evidence of dysmotility. None of these 18 patients had increased dysphagia postoperatively; 8 of 18 reported improvement with swallowing. Five patients in the selective group (0.8%) had persistent postoperative dysphagia caused by technical error (four patients) or with no identifiable cause (one patient). The estimated charge or collection reduction with use of the selective evaluation was 1,253,100 US dollars or 395,000 US dollars, respectively. CONCLUSIONS: Selective use of manometry and pH monitoring was cost effective and safe in this series. Although esophageal manometry and 24-hour pH monitoring might be necessary with abnormal findings on videofluoroscopy or atypical symptoms, in our experience, their routine use is not essential in preoperative evaluation of patients undergoing fundoplication for gastroesophageal reflux disease.     

"Infectious tolerance" develops after the spontaneous acceptance of Lewis-to-Dark Agouti rat liver transplants

BACKGROUND: After monoclonal antibody or donor-specific transfusion treatment, infectious tolerance to rat or mouse heart or skin transplants can be passed on to naive recipients by adoptive transfer of tolerant lymphocytes. We examined whether similar regulatory cells develop after the spontaneous acceptance of Lewis-to-Dark Agouti (DA) rat liver transplants without immunomodulating agents. METHODS: After Lewis-to-DA rat liver transplantation, 100 x 10(6) splenocytes were harvested and adoptively transferred into a 450 rad-irradiated naive DA rat 24 hours before Lewis heart transplantation. Adoptive transfer of CD4+ or CD8+ T cells was also examined. In some experiments, splenocytes from recipients with long-term accepted Lewis hearts induced by adoptive transfer were serially transferred to multiple generations of recipients before Lewis rat heart transplantation. In vitro mixed lymphocyte culture response and cytotoxic T lymphocyte generation were measured. RESULTS: When splenocytes from a DA rat recipient >60 days after Lewis rat liver acceptance were transferred into irradiated DA rat recipients, all Lewis rat hearts were accepted, whereas third-party Brown-Norway rat hearts were rejected. However, splenocytes from DA rat recipients 30 days after liver transplantation did not prolong Lewis rat heart survival. Adoptive transfer of 40 x 10(6) CD4+, 10 x 10(6) CD4+ or 10 x 10(6) CD8+ cells from a DA rat bearing Lewis rat liver >60 days resulted in acceptance of 88%, 80%, or 57% acceptance of Lewis rat hearts, respectively. Serial second and third adoptive transfer of long-term survivor splenocytes resulted in the acceptance of all Lewis rat hearts. In mixed lymphocyte culture, splenocytes from a naive DA rat and a DA rat accepting a Lewis rat liver transplant for >60 days showed similar proliferative responses to both Lewis and Brown-Norway rat stimulators. An equivalent level of indirect cytotoxic T lymphocyte activity was exhibited by splenocytes from both a naive DA and a DA rat accepting a Lewis rat liver transplant for >60 days. CONCLUSIONS: Regulatory cells developing after the spontaneous acceptance of a Lewis to DA liver transplant can serially adoptively transfer the acceptance of a Lewis rat cardiac graft in spite of the presence of in vitro antidonor reactivity. Both CD4+ and CD8+ populations have this regulatory activity, although the CD4+ population plays the dominant role.