Prehospital rapid sequence induction by emergency physicians: is it safe?

OBJECTIVES: To determine if there were differences in practice or intubation mishap rate between anaesthetists and accident and emergency physicians performing rapid sequence induction of anaesthesia (RSI) in the prehospital setting. METHODS: All patients who underwent RSI by a Helicopter Emergency Medical Service (HEMS) doctor from 1 May 1997 to 30 April 1999 were studied by retrospective analysis of in-flight run sheets. Intubation mishaps were classified as repeat attempts at intubation, repeat drug administration and failed intubation. RESULTS: RSI was performed on 359 patients by 10 anaesthetists (202 patients) and nine emergency physicians (157 patients). Emergency physicians recorded a larger number of patients as having Cormack and Lehane grade 3 or 4 laryngoscopy than anaesthetists (p<0.0001) but were less likely to use a gum elastic bougie to assist intubation (p=0.024). Patients treated by emergency physicians did not have a significantly different pulse, blood pressure, oxygen saturation or end tidal CO2 to patients treated by anaesthetists at any time after intubation. Emergency physicians were more likely to anaesthetise patients with a Glasgow Coma Score >12 than anaesthetists (p=0.003). There were two failed intubations (1%) in the anaesthetist group and four (2.5%) in the emergency physician group. Repeat attempts at intubation and repeat drug administration occurred in <2% of each group. CONCLUSIONS: RSI performed by emergency physicians was not associated with a significantly higher failure rate or an increased number of intubation mishaps than RSI performed by anaesthetists. Emergency physicians were able to safely administer sedative and neuromuscular blocking drugs in the prehospital situation. It is suggested that emergency physicians can safely perform rapid sequence induction of anaesthesia and intubation.

Mineral density and bone strength are dissociated in long bones of rat osteopetrotic mutations.

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.

Endochondral bone formation in toothless (osteopetrotic) rats: failures of chondrocyte patterning and type X collagen expression.

The pacemaker of endochondral bone growth is cell division and hypertrophy of chondrocytes. The developmental stages of chondrocytes, characterized by the expression of collagen types II and X, are arranged in arrays across the growth zone. Mutations in collagen II and X genes as well as the absence of their gene products lead to different, altered patterns of chondrocyte stages which remain aligned across the growth plate (GP). Here we analyze GP of rats bearing the mutation toothless (tl) which, apart from bone defects, develop a progressive, severe chondrodystrophy during postnatal weeks 3 to 6. Mutant GP exhibited disorganized, non-aligned chondrocytes and mineralized metaphyseal bone but without cartilage mineralization or cartilaginous extensions into the metaphysis. Expression of mRNA coding for collagen types II (Col II) and X (Col X) was examined in the tibial GP by in situ hybridization. Mutant rats at 2 weeks exhibited Col II RNA expression and some hypertrophied chondrocytes (HC) but no Col X RNA was detected. By 3rd week, HC had largely disappeared from the central part of the mutant GP and Col II RNA expression was present but weak and in 2 separate bands. Peripherally the GP contained HC but without Col X RNA expression. This abnormal pattern was exacerbated by the fourth week. Bone mineralized but cartilage in the GP did not. These data suggest that the tl mutation involves a regulatory function for chondrocyte maturation, including Col X RNA synthesis and mineralization, and that the GP abnormalities are related to the Col X deficiency. The differences in patterning in the tl rat GP compared to direct Col X mutations may be explained by compensatory effects.

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat.

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.

The toothless osteopetrotic rat has a normal vitamin D-binding protein-macrophage activating factor (DBP-MAF) cascade and chondrodysplasia resistant to treatments with colony stimulating factor-1 (CSF-1) and/or DBP-MAF.

The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in this mutation is unaffected by either molecule. Thus, the tl mutation intercepts the function of a gene required for both normal endochondral ossification and bone resorption, thereby uncoupling the coordination of skeletal metabolism required for normal long bone growth.

Facial development and type III collagen RNA expression: concurrent repression in the osteopetrotic (Toothless,tl) rat and rescue after treatment with colony-stimulating factor-1.

The toothless (osteopetrotic) mutation in the rat is characterized by retarded development of the anterior facial skeleton. Growth of the anterior face in rats occurs at the premaxillary-maxillary suture (PMMS). To identify potential mechanisms for stunted facial growth in this mutation we compared the temporospatial expression of collagen I (Col I) and collagen III (Col III) RNA around this suture in toothless (tl) rats and normal littermates by in situ hybridization of specific riboprobes in sagittal sections of the head. In normal rats, the suture is S shaped at birth and becomes highly convoluted by 10 days with cells in the center (fibroblasts and osteoblast progenitors) expressing Col III RNA and those at the periphery (osteoblasts) expressing no Col III RNA but high amounts of Col I RNA throughout the growth phase (the first 2 postnatal weeks). In the mutant PMMS, cells were reduced in number, less differentiated, and fewer osteoblasts were encountered. Expression of Col I RNA was at normal levels, but centrosutural cells expressed Col III RNA only after day 6 and then only weakly. A highly convoluted sutural shape was never achieved in mutants during the first 2 postnatal weeks. Treatment of tl rats with the cytokine CSF-1 improved facial growth and restored cellular diversity and Col III RNA expression in the PMMS to normal levels. Taken together, these data suggest that normal facial growth in rats is related to expression of Col III RNAby osteoblast precursors in the PMMS, that these cells are deficient in the tl mutation and are rescued following treatment with CSF-1.

Auditory ossicle abnormalities and hearing loss in the toothless (osteopetrotic) mutation in the rat and their improvement after treatment with colony-stimulating factor-1.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat.

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

The effects of colony-stimulating factor-1 on the number and ultrastructure of osteoclasts in toothless (tl) rats and osteopetrotic (op) mice.

The role of colony-stimulating factor-1 (CSF-1 or M-CSF) in osteoclast development is illustrated by observations that administration of exogenous CSF-1 increases osteoclast number and improves the skeletal sclerosis of two osteopetrotic mutations, toothless (tl) in the rat and osteopetrotic (op) in the mouse. We examined the effects of CSF-1 treatment on the number and ultrastructure of osteoclasts in the tibial metaphysis of normal and mutant animals of both stocks to understand the similarities and differences between these two mutations. Osteoclasts from normal animals of both stocks were abundant and possessed the ultrastructural features of active cells. These included apical areas in contact with mineralized surfaces with tightly apposed clear zones, extensive ruffled borders, and a vacuolated cytoplasm with numerous mitochondria. In toothless rats osteoclasts were difficult to locate and those present had poorly defined ruffled borders, fewer cytoplasmic vacuoles, and a basal membrane with both smooth and ruffled areas. Large lipid accumulations were often found near tl osteoclasts. Osteoclasts in op mice were difficult to find, but more numerous than in tl rats. Unlike tl osteoclasts, those of op mice possessed very well developed ruffled borders, small clear zones, and large electron-dense cytoplasmic inclusions. These cells also had unusual basal membranes with both smooth and ruffled regions. CSF-1 treatment increased the number of osteoclasts in both mutant stocks, normalizing the numbers in op mice, but not tl rats. CSF-1 injections caused dramatic changes in the morphology of tl osteoclasts, including increased incidence and size of ruffled borders and cytoplasmic vacuolization. The growth factor had little effect on ruffled borders or clear zones in op mice. Interestingly, mutant osteoclasts of both stocks exhibited a ruffled basal membrane in response to CSF-1 treatment. This increase in membrane ruffling may reflect the ability of CSF-1 to promote rapid formation of osteoclasts from mononuclear precursors in a more permissive microenvironment. Our data indicate that CSF-1 is not required for the development of at least some osteoclasts. The differences in response to CSF-1 treatment which we report lead us to speculate that additional factors may be involved in osteoclastogenesis.

Local anaesthetic for lower-limb revascularization in high-risk patients.

BACKGROUND: A few patients with critical limb ischaemia are believed to be too unfit for an attempt at revascularization using conventional anaesthesia. METHODS: A retrospective analysis was undertaken of 46 revascularization procedures performed in high-risk patients for critical limb ischaemia between 1989 and 1995, in which local anaesthetic techniques were utilized in preference to general or spinal anaesthesia. RESULTS: Cumulative survival rates at 6, 12 and 24 months were 67, 57 and 51 per cent. Primary patency rates were 77 per cent at 6 months, 67 per cent at 12 months and 53 per cent at 24 months, with associated limb salvage rates of 87, 87 and 79 per cent. CONCLUSION: Selective use of local anaesthetic techniques extends the benefits of limb salvage to patients considered unfit for conventional anaesthesia.

Axillary brachial plexus block--an underused technique in the accident and emergency department.

OBJECTIVE: To compare axillary brachial plexus block and Bier's block as methods of providing upper limb anaesthesia. METHODS: Axillary brachial plexus or Bier's blocks were performed on all patients requiring upper limb anaesthesia in a three month period. For Bier's block, a single cuff tourniquet and 3 mg/kg 0.5% prilocaine were used. For axillary plexus block, 40 ml 1% lignocaine with adrenaline (1:200,000) were used, given by perivascular or transarterial technique. Prospective analysis was made of time to complete limb anaesthesia, type of procedure performed, and duration of limb anaesthesia. Patient perception of analgesia and satisfaction with the method of anaesthesia was assessed using a 10 point visual analogue scale. RESULTS: 75 patients underwent procedures requiring upper limb anaesthesia; 39 received axillary plexus block and 36 Bier's block. 72% of Bier's blocks and 77% of axillary plexus provided complete anaesthesia without the need for supplemental analgesia. The median time to onset of anaesthesia was 10 min for Bier's block and 32.5 min for axillary block (P < 0.001). The median duration of anaesthesia was 15 min for Bier's block and 240 min for axillary block (P < 0.001). Mean scores for analgesia were 9.7 for axillary blocks and 8.8 for Bier's block (P < 0.001). 87% of the axillary block group were completely satisfied with the method of anaesthesia, compared with 56% of the Bier's block group. CONCLUSIONS: Brachial plexus blocks are an alternative form of providing upper limb anaesthesia in the accident and emergency department. They are relatively simple to perform, well tolerated by patients, and have the advantage of providing prolonged analgesia without the need for additional medication.

Ultrastructural evidence of abnormally short and maldistributed actin stress fibers in osteopetrotic (toothless) rat osteoblasts in situ after detergent perfusion.

Osteopetrosis is a heterogenous group of metabolic bone diseases characterized by a generalized increase in skeletal mass, the product of reduced bone resorption and interceptions in the development and/or function of osteoclasts. In one such mutation in the rat, toothless (tl), osteoblasts are absent from older bone surfaces and there is evidence for aberrant osteoblast gene expression and function. Given the emerging appreciation of the role of osteoblasts in the differentiation and activation of osteoclasts, we have examined ultrastructural features of the cytoskeleton of normal and mutant osteoblasts after perfusion fixation with the non-ionic detergent Triton X-100. This procedure reduces the electron density of the cytoplasm, rendering visible the microfilamentous network in osteoblasts. In normal osteoblasts a prominent system of stress fibers (bundles of actin microfilaments) run parallel to the cell membrane adjacent to osteoid surfaces, stretching for 75% of that distance. However, only 50% of mutant (tl) osteoblasts had stress fibers and in these cells stress fibers were either significantly shorter (18% of normal) or distributed intracellularly rather than along the osteoid surface. In mutant osteoblasts without stress fibers, 20% showed ultrastructural signs of cell degeneration. Given the role of stress fibers in cellular attachment ot extracellular matrices, these observations suggest that the reduced number of osteoblasts in tl rats may be related to their inability to organize actin filaments and adhesion plaques for attachment to bone surfaces. We propose that a feature of osteopetrosis in the tl rat is a disruption of the mechanisms that regulate the synthesis, sorting, and/or assembly of actin.

Thrombocytopenia in the toothless (osteopetrotic) rat and its rescue by treatment with colony-stimulating factor-1.

Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover.

Dense incisors (din): a new mouse mutation on chromosome 16 affecting tooth eruption and body size.

Dense incisors (din) is a new autosomal recessive mutation in the mouse that interferes with complete eruption of the incisors. The initial eruption of incisors through the gingiva does not differ in mutants and normal littermates, but subsequent further eruption of incisors is arrested in mutants. Radiographic examinations show that, because the incisors do not erupt, continued dentin formation gradually occludes the pulp chambers of these teeth creating as dense incisor. The arrested eruption of the incisor results in an anterior open bite. The pleiotropic phenotype of din/din mutant mice also includes small body size, reduced ear pinna size, and coat color dilution. The din mutation was mapped to Chr 16 near the pituitary transcription factor gene Pit1, but din is not a mutation in Pit1.

Spontaneously hypertensive rats develop pulmonary hypertension and hypertrophy of pulmonary venous sphincters.

This study explored the spontaneously hypertensive rat as an animal model of pulmonary hypertension and sought to identify anatomic changes in its pulmonary microvasculature, especially focal constrictions of pulmonary veins (sphincters). The average systemic and pulmonary artery blood pressures were 172/139 (+/- 9/9) and 36/14 (+/- 4/3), respectively, for spontaneously hypertensive Wistar Kyoto rats (SHR), and 134/83 (+/- 8/2) and 20/10 (+/- 2/2) for normotensive Wistar Kyoto rats (WKY) (P < 0.01 for both). Light microscopy of the lungs in SHR showed muscularization of both arteries and veins, but this was more pronounced in the small pulmonary veins. Perivascular edema was also present. There were 20 (+/- 4) leukocytes per 100 microns of capillary length in SHR and 9 (+/- 2) in WKY (P < 0.001). Transmission electron microscopy showed focal venous smooth muscle was greater in SHR than in WKY. Scanning electron microscopy of vascular casts showed the average maximal focal venous contraction (sphincter) was 54% (+/- 10) of its diameter in SHR, but was only 6% (+/- 4) in WKY (P < 0.01). Arterial contraction occurred in the hypertensive rats as bourglass narrowings of the casts, but was less conspicuous than venous constrictions. The mean alveolar capillary diameter was 8.1 microns (+/- 1.6) in SHR, compared with 6.3 microns (+/- 1.0) in WKY (P < 0.01). The central interspace between capillaries was 3.2 microns (+/- 1.6) in SHR and 6.0 microns (+/- 3.6) in WKY (P < 0.01). The venous contraction, capillary size, and capillary interspace distance correlated with the pulmonary blood pressure. The spontaneously hypertensive rat can be a model of pulmonary hypertension with its most notable structural change being increased muscularity in the small pulmonary veins.

Decreased growth hormone receptor expression in long bones from toothless (osteopetrotic) rats and restoration by treatment with colony-stimulating factor-1.

Growth hormone (GH) is known to regulate growth and development of skeletal tissues. This study examined the distribution of growth hormone receptor (GHR) expression in tibias from normal and osteopetrotic tl/tl rats. For normal 2 week-old rats, GHR expression was detected immunocytochemically in cells of the articular and epiphyseal cartilage, primary and secondary ossification centres, zone of resting cartilage and bone marrow. Within the marrow, GHR immunopositive cells were concentrated in the central cone and largely excluded from the zone of immature progenitors at the periphery. For the marrow haemopoietic compartment, GHR expression was almost restricted to the nucleus in large mononuclear cells, adipocytes and megakaryocytes. A population of small lymphocytelike cells in the marrow periphery expressed GHR on the plasma membrane. GHR was not detected in mature erythroid cells, macrophages, granulocytes, or osteoclasts. The expression of GHR was significantly reduced in bone marrow cells of the tl/tl rat (p < 0.001) compared with normal animals. Injection of recombinant CSF-1 into tl/tl rats every 48 hours for 2 weeks from birth restored GHR-positive cells to the central core of the marrow space. The most striking change was the appearance of substantial numbers of mononuclear cells expressing abundant GHR on the cell surface. We infer that these cells are a novel subset of CSF-1 responsive cells involved in bone resorption. The differences in relative expression of GHR by bone marrow cells in untreated and CSF-1-treated tl/tl rats suggests a CSF-1-dependent recruitment of cells bearing surface GHRs.

Normalization of mineral homeostasis after reversal of osteopetrosis.

Whether a radiographic and histologic cure of osteopetrosis includes normalization of mineral homeostasis remains unknown. Thus, we explored the extent of defective mineral metabolism in the microphthalmic (mi/mi) mouse before and after cure. Under basal conditions mi mutants exhibit normocalcemia, hypophosphatemia, and elevated renal 25-hydroxyvitamin D-1-hydroxylase activity. However, administration of PTHrP (3 micrograms/h x 24 h) further stimulated enzyme activity in mi mutants with active disease, to a level no different than that in treated normals. Serum phosphorus levels also declined in mi/mi mice following PTHrP, suggesting a normal renal response to this hormone. In contrast, failure to suppress enzyme function in mi/mi mice following prolonged calcitriol infusion indicates that the observed enhancement of 1,25-dihydroxyvitamin D production occurred secondary to autonomous parathyroid function and/or nonparathyroid hormone-related stimuli. Although an increased fractional excretion and decreased tubular reabsorption of phosphate were demonstrated in mi/mi mice, serum PTH levels were no different in mi mutants compared with normal littermates. Following skeletal cure, the mi/mi mice surprisingly display normal serum phosphorus levels and renal enzyme activity. Moreover, treatment restored normal responsiveness to calcitriol suppression and maintained normal PTHrP responsiveness of enzyme activity. These data indicate that the cure of osteopetrosis in the mi mutant is universal and includes normalization of serum phosphorus and renal 25-hydroxyvitamin D-1-hydroxylase. Furthermore, these data suggest that phosphate depletion of unknown origin is the likely cause of elevated enzyme activity in this murine osteopetrotic mutant.

Microvascular pattern in the metaphysis during bone growth.

BACKGROUND: Little is known about the three-dimensional micromorphology of vessels in the growth zone of long bones, where significant vasculogenesis occurs. Therefore, we examined the microvascular pattern of the femoral metaphysis. METHODS: Six-week-old normal rats of either sex were used. We cast the femurs of 14 rats with Mercox for scanning electron microscopy (SEM), and in 10 rats we prepared tissue sections of femurs for light (LM) and transmission electron microscopy (TEM). RESULTS: In the LM, calcified cartilage was found to define cylindrical compartments beneath the last row of hypertrophied chondrocytes of the metaphyseal growth plate. These compartments ran in the bone's longitudinal axis and contained a single capillary profile. Endothelial cells of these capillaries often showed increased cytoplasmic volume and loose texture of nuclear chromatin. Cast metaphyses by SEM showed numerous parallel vascular loops with nodular protrusions 10-12 microns in diameter at their tips. The loops had ascending and descending limbs with a luminal diameter of 10-14 microns. Small projections 4-5 microns in diameter and delicate crests were sometimes found on the tip of the larger nodes. In a 100 x 100 microns area, there were 14-17 large nodes. By TEM, capillary sprouts were identified at the level beneath the last row of hypertrophied chondrocytes. These capillaries had voluminous endothelial cells rich in free ribosomes and rough endoplasmic reticulum. Endothelial cell nuclei were rounded and showed loose chromatin texture. Endothelial cells were connected by intermediate junctions and there was no basal lamina. Deeper into the metaphysis, arterioles and sinusoids were present. CONCLUSIONS: We conclude that the metaphyseal plate of the growing rat offers an optimal model to study vasculogenesis. Capillary sprouts can be readily identified, measured, and counted because they are located within a plane bordering against avascular cartilage. In addition, by using microvascular corrosion casting in SEM not only capillary sprouting per se but also different stages of neovascularization, indicated by differently sized nodular projections at the tip of vascular loops, can be studied in the growing long bone.

CSF-1 treatment promotes angiogenesis in the metaphysis of osteopetrotic (toothless, tl) rats.

It has recently been shown that following treatment with colony-stimulating factor-1 (CSF-1) the osteopetrotic condition in toothless (tl) rats greatly improves and growth is accelerated. We have examined the effects of such treatment on the microvasculature of the distal femoral chondro-osseous junction, a site where bone growth in length is coordinated with angiogenesis. Vascular casts and ultrastructural analyses of this region showed that, compared to untreated normal rats, untreated mutants showed little bone growth or angiogenesis. When mutants were treated with CSF-1 angiogenesis was markedly accelerated. These data show a remarkable effect of this growth factor on angiogenesis in this osteopetrotic mutation. Whether this effect of CSF-1 on angiogenesis is direct or indirect is not known and indicates that its effects on the normal microvasculature deserve further study.

Abnormalities of phosphoprotein gene expression in three osteopetrotic rat mutations: elevated mRNA transcripts, protein synthesis, and accumulation in bone of mutant animals.

Osteoclast abnormalities that characterize osteopetrosis, a disorder of bone resorption, may derive from aberrant signals from the osteoblast or the bone matrix. In the present studies, both synthesis and the bone matrix content of the major bone phosphoprotein component, osteopontin, were found to be elevated in three osteopetrotic rat mutations (ia, op, and tl). In whole bone, a twofold increase in the content of the characteristic amino acid O-phosphoserine for osteopontin occurred in op and tl mutant long bone, but a smaller (15%) and more variable increase was observed in ia mutant rat long bone. Extraction of the bone matrix components and partial purification by reverse phase chromatography showed a twofold increase in a phosphoprotein fraction relative to other noncollagenous components. Amino acid analysis and staining characteristics of SDS-PAGE fractionated proteins indicated this to be osteopontin. Organ cultures of calvarial bone from 4 day ia osteopetrotic mutant and normal rats in the presence of 3H-proline showed increased synthesis of this 60 kD protein, which was stimulated by vitamin D. Preparation of total cellular RNA from bone of 2- and 6-week-old mutants and normal rats supported increased synthesis of osteopontin as reflected by hybridization with osteopontin cDNA probe, showing significantly higher levels of mRNA transcripts in ia (3-5 fold), tl (1.4-2 fold), and op (6-25 fold) mutant bone compared to normal littermates. The changes in osteopontin mRNA levels in mutant bone were also examined in relation to other growth and phenotype-expressed genes. The findings of increased accumulation of osteopontin in osteopetrotic bone and increased synthesis by osteoblasts are interesting in light of the previously reported decrease in bone osteocalcin content (Endocrinology, 126:966, 1990), confirmed here by decreased osteocalcin mRNA transcripts. Such aberrations in the composition of skeletal extracellular matrix could be a reflection of or a contributing factor to the osteoclast abnormalities of some of these osteopetrotic disorders.