Surgical protocol involving the infusion of paramagnetic microparticles for preferential incorporation within porcine islets.

INTRODUCTION: Despite significant advances, widespread applicability of islet cell transplantation remains elusive. Refinement of current islet isolation protocols may improve transplant outcomes. Islet purification by magnetic separation has shown early promise. However, surgical protocols must be optimized to maximize the incorporation of paramagnetic microparticles (MP) within a greater number of islets. This study explores the impact of MP concentration and infusion method on optimizing MP incorporation within islets. METHODS: Five porcine pancreata were procured from donors after cardiac death. Splenic lobes were isolated and infused with varying concentrations of MP (8, 16, and 32 × 10(8) MP/L of cold preservation solution) and using one of two delivery techniques (hanging bag versus hand-syringe). After procurement and infusion, pancreata were stored at 0°C to 4°C during transportation (less than 1 hour), fixed in 10% buffered formalin, and examined by standard magnetic resonance imaging (MRI) and histopathology. RESULTS: T2*-weighted MRI showed homogeneous distribution of MP in all experimental splenic lobes. In addition, histologic analysis confirmed that MP were primarily located within the microvasculature of islets (82% to 85%), with few MP present in acinar tissue (15% to 18%), with an average of five to seven MP per islet (within a 5-µm thick section). The highest MP incorporation was achieved at a concentration of 16 × 10(8) MP/L using the hand-syringe technique. CONCLUSION: This preliminary study suggests that optimization of a surgical protocol, MP concentrations, and applied infusion pressures may enable more uniform distribution of MP in the porcine pancreas and better control of MP incorporation within islets. These results may have implications in maximizing the efficacy of islet purification by magnetic separation.

Pancreas oxygen persufflation increases ATP levels as shown by nuclear magnetic resonance.

BACKGROUND: Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS: Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS: Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION: The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

Persufflation improves pancreas preservation when compared with the two-layer method.

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Continuous real-time viability assessment of kidneys based on oxygen consumption.

BACKGROUND: Current ex vivo quality assessment of donor kidneys is limited to vascular resistance measurements and histological analysis. New techniques for the assessment of organ quality before transplantation may further improve clinical outcomes while expanding the depleted deceased-donor pool. We propose the measurement of whole organ oxygen consumption rate (WOOCR) as a method to assess the quality of kidneys in real time before transplantation. METHODS: Five porcine kidneys were procured using a donation after cardiac death (DCD) model. The renal artery and renal vein were cannulated and the kidney connected to a custom-made hypothermic machine perfusion (HMP) system equipped with an inline oxygenator and fiber-optic oxygen sensors. Kidneys were perfused at 8 degrees C, and the perfusion parameters and partial oxygen pressures (pO(2)) were measured to calculate WOOCR. RESULTS: Without an inline oxygenator, the pO(2) of the perfusion solution at the arterial inlet and venous outlet diminished to near 0 within minutes. However, once adequate oxygenation was provided, a significant pO(2) difference was observed and used to calculate the WOOCR. The WOOCR was consistently measured from presumably healthy kidneys, and results suggest that it can be used to differentiate between healthy and purposely damaged organs. CONCLUSIONS: Custom-made HMP systems equipped with an oxygenator and inline oxygen sensors can be applied for WOOCR measurements. We suggest that WOOCR is a promising approach for the real-time quality assessment of kidneys and other organs during preservation before transplantation.

Moderate alcohol consumption suppresses bone turnover in adult female rats.

Chronic alcohol abuse is a major risk factor for osteoporosis but the effects of moderate drinking on bone metabolism are largely uninvestigated. Here, we studied the long-term dose-response (0, 3, 6, 13, and 35% caloric intake) effects of alcohol on cancellous bone in the proximal tibia of 8-month-old female rats. After 4 months of treatment, all alcohol-consuming groups of rats had decreased bone turnover. The inhibitory effects of alcohol on bone formation were dose dependent. A reduction in osteoclast number occurred at the lowest level of consumption but there were no further reductions with higher levels of consumption. An imbalance between bone formation and bone resorption at higher levels of consumption of alcohol resulted in trabecular thinning. Our observations in rats raise the concern that moderate consumption of alcoholic beverages in humans may reduce bone turnover and potentially have detrimental effects on the skeleton.

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression.

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Estrogen has rapid tissue-specific effects on rat bone.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1-32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.

Effects of ethanol on gene expression in rat bone: transient dose-dependent changes in mRNA levels for matrix proteins, skeletal growth factors, and cytokines are followed by reductions in bone formation.

Several studies were performed in female rats to determine dose and time course changes in mRNA levels for matrix proteins in bone after a single administration of ethanol. As expected, dose-dependent transient increases in blood ethanol were measured. Additionally, there was mild hypocalcemia with no change in immunoreactive parathyroid hormone. Coordinated dose-dependent increases in mRNA for type 1 collagen, osteonectin, and osteocalcin were noted in the proximal tibial metaphysis 6 hr after ethanol was given, with the peak values occurring at a dose of 1.2 g/kg (0.4 ml). Similar increases in mRNA levels for matrix proteins were noted in lumbar vertebrae after ethanol treatment. The changes were specific for bone; ethanol had no effect on mRNA levels for matrix proteins in the uterus or liver, although the mRNA concentrations tended to be reduced in uterus. Message levels for several cytokines implicated in the regulation of bone turnover were also assayed; mRNA levels for transforming growth factor-beta1, transforming growth factor-beta2, interferon-gamma, and interleukin-6 were unchanged at doses ranging from 0.14 to 1.7 g/kg. At the highest dose of ethanol, the mRNA level for tumor necrosis factor-alpha was elevated while the level for insulin-like growth factor-1 was reduced. The time course effects of ethanol (0.4 ml dose) were determined in a separate experiment. Ethanol resulted in a transient increase in mRNA levels for the three bone matrix proteins assayed. However, matrix protein synthesis, as determined by incorporation of 3H-proline into the proximal tibial metaphysis, was not changed after 6 hr. The changes in mRNA levels for the matrix proteins were preceded by brief, transient decreases in mRNA levels for interleukin-1beta, interferon-gamma, and migration inhibitory factor, and followed by a more prolonged decrease in the mRNA level for insulin-like growth factor-1. A subsequent study was performed to determine the effects of repetitive daily treatment with ethanol on rat bone. After 7 days, there were highly significant decreases in the mRNA level for type 1 collagen, as well as decreased bone formation. These results suggest that ethanol may alter bone metabolism by disturbing signal transduction pathways that regulate the expression of genes for bone matrix proteins, skeletal growth factors, and cytokines.

Dietary ethanol does not accelerate bone loss in ovariectomized rats.

The abuse of alcohol is a behavior that can significantly compromise skeletal health. Because postmenopausal women are already at risk for low bone mass and osteoporotic fracture, this investigation sought to determine whether high concentrations of dietary ethanol exacerbate the bone loss associated with ovariectomy in rats, an animal model of human postmenopause. Six-month-old Sprague-Dawley rats were ovariectomized or sham-operated and randomly divided into groups fed a modified Lieber-DeCarli liquid diet isocalorically supplemented with 0%, 13%, or 35% ethanol (by daily caloric intake), for a period of 2 months. All animals were injected with fluorochromes at the start, 2 weeks, and 2 days before sacrifice to label mineralizing bone surfaces. At sacrifice, blood, uterus, and tibiae were harvested. No differences in serum calcium or cholesterol were found. Serum creatinine was also found to be unvaried, indicating this level of alcohol consumption did not compromise liver function. Dietary alcohol consumption at 35% of daily caloric intake was determined to increase tibial cortical medullary area and endocortical perimeter, while not affecting cortical area and periosteal perimeter. Ovariectomy significantly increased indices of bone turnover and resulted in cancellous bone loss, whereas alcohol consumption had no additional detrimental effects. This was a consistent pattern for other indices of proximal tibial architecture. In summary, this investigation has found that chronic ingestion of high concentrations of alcohol does not accentuate bone loss in ovarian hormone-deficient adult female rats.

Effects of growth hormone and low dose estrogen on bone growth and turnover in long bones of hypophysectomized rats.

Pituitary hormones are recognized as critical to longitudinal growth, but their role in the radial growth of bone and in maintaining cancellous bone balance are less clear. This investigation examines the histomorphometric effects of hypophysectomy (Hx) and ovariectomy (OVX) and the subsequent replacement of growth hormone (GH) and estrogen (E), in order to determine the effects and possible interactions between these two hormones on cortical and cancellous bone growth and turnover. The replacement of estrogen is of interest since Hx results in both pituitary and gonadal hormone insufficiencies, with the latter being caused by the Hx-associated reduction in follicle stimulating hormone (FSH). All hypophysectomized animals received daily supplements of hydrocortisone (500 microg/kg) and L-thyroxine (10 microg/kg), whereas intact animals received daily saline injections. One week following surgery, hypophysectomized animals received either daily injections of low-dose 17 beta-estradiol (4.8 microg/kg s.c.), 3 X/d recombinant human GH (2 U/kg s.c.), both, or saline for a period of two weeks. Flurochromes were administered at weekly intervals to label bone matrix undergoing mineralization. Whereas Hx resulted in reductions in body weight, uterine weight, and tibial length, OVX significantly increased body weight and tibial length, while reducing uterine weight. The combination of OVX and Hx resulted in values similar to Hx alone. Treatment with GH normalized body weight and bone length, while not affecting uterine weight in hypophysectomized animals. Estrogen increased uterine weight, while not impacting longitudinal bone growth and reduced body weight. Hypophysectomy diminished tibial cortical bone area through reductions in both mineral appositional rate (MAR) and bone formation rate (BFR). While E had no effect, GH increased both MAR and BFR, though not to sham-operated (control) levels. Hypophysectomy reduced proximal tibial trabecular number and cancellous bone area, and increased trabecular separation. Both GH and E reduced cancellous osteopenia, although employing different mechanisms. GH reduced the decrease in trabecular thickness, whereas E reduced the decrease in trabecular number and the increase in trabecular separation. Hypophysectomy reduced both Tb.MAR and Tb.BFR while treatment with GH enhanced them. This investigation has shown that Hx and GH have a dramatic impact on selected static and dynamic indices of rat cortical and cancellous histomorphometry. Furthermore, the mechanisms of action of GH and E differ, and suggest that some of the skeletal changes associated with Hx are caused by deficiencies in estrogen as well as deficiencies in growth hormone.

Effects of aluminum on rat bone cell populations.

Aluminum (Al) loading is associated with reduced bone formation and osteomalacia in human and certain animal models. However, uncertainty exists as to the cellular effect(s) of Al as both inhibition and stimulation of osteoblast proliferation have been reported. Furthermore, the extent to which Al affects osteoprogenitor cell populations is unknown. To determine the cellular effects of Al in the rat, an animal model in which Al bone disease has been produced, we compared the in vitro effect of 10-50 microns Al on the proliferation and hydroxyproline collagen formation of marrow osteoprogenitor stromal cell populations and perinatal rat calvarial osteoblasts. In subconfluent cultures, Al suppressed proliferation of both marrow fibroblast-like stromal cells and calvarial osteoblasts. In confluent cultures, however, Al selectively stimulated periosteal fibroblast and osteoblast DNA synthesis and collagen (hydroxyproline) production, both in the presence or absence of 1,25-dihydroxyvitamin D. Osteocalcin was not detected in osteoblast-conditioned media or extracellular matrix. These observations suggest that the bone formation defect associated with Al toxicity in growing rats may be a function of impaired patterns of osteoprogenitor/osteoblast proliferation. Furthermore, the Al-stimulated increase in collagen formation is consistent with the development of osteomalacia in Al-toxic humans and animals. The mechanism by which Al stimulated DNA synthesis and collagen production in more mature cultures awaits further study.

Rat tail suspension causes a decline in insulin receptors.

Decreased muscular activity results in weakness and muscular atrophy. Coincident with this protein catabolic state is glucose intolerance and hyperinsulinemia. Rats were tail suspended for 7 to 14 days to accomplish unloading of the hindlimbs. Insulin resistance was documented in these animals by a 14 day tail suspension-related 26% increase in serum glucose in spite of a 253% increase in serum insulin concentration. Microsomal membranes were prepared from hindlimb muscles and specific binding of insulin and insulin-like growth factor I (IGF-I) were determined in these membranes. Insulin binding was decreased by 27% at 7 days and by 21% at 14 days. In contrast, IGF-I binding was unchanged at 7 days and was increased by 24% at 14 days. Liver membrane insulin receptors also had declined by 14 days of suspension, suggesting that the change in insulin receptors was a generalized, humorally-mediated phenomenon. These data suggest that tail suspension in rats results in insulin resistance, hyperinsulinemia, a decline in insulin receptors in liver and muscle, and a relative increase in muscle membrane IGF-I receptors. These data are consistent with the hypothesis that resistance to insulin's effects on protein metabolism in skeletal muscle may contribute to the protein catabolism associated with decreased muscular activity.

Skeletal effects of sodium fluoride during hypokinesia.

This study tested the capacity of fluoride (F) to prevent the disuse-associated reduction in bone formation/growth. Suspending young male Wistar rats by the tail for 2-2.5 weeks reduced femoral cortical (P less than 0.05) and trabecular (P less than 0.01) bone areas. Tetracycline labelling showed that the decrement in cortical area was largely due to a reduction in the percent periosteal mineralizing surface (PsMS). Periosteal mineral apposition rate (PsMAR) was not affected. Endosteal mineralizing surface (EsMS) and mineral apposition rate (EsMAR) were significantly stimulated spontaneously during the second week of suspension. F treatment (5 mg/kg/day i.p.) prevented the loss in bone area, and established a trend toward increased PsMS without affecting EsMS and EsMAR. None of these changes are associated with alterations in serum Ca, P or osteocalcin. F treatment in hypokinetic animals caused a decrease in serum PTH (-21% compared to control; P = 0.001). We conclude that F prevents the development of hypokinetic osteopenia in rats.