Cytoskeletal-induced alterations in the adhesion of HT-1080 fibrosarcoma cells to extracellular matrix.

We have investigated the adhesion of the human fibrosarcoma cell line, HT-1080, transfected with glial fibrillary acidic protein (GFAP) to a variety of extracellular matrix macromolecules (ECM) including collagen type IV, laminin, and fibronectin. The GFAP-transfectants demonstrated altered adhesiveness to extracellular matrix substrates when compared to controls. GFAP-positive, heavy metal-induced fibrosarcoma cells were more adherent to plastic and collagen type IV than were the parental or uninduced cells. In contrast, GFAP-positive fibrosarcoma cells were less adherent to laminin- or fibronectin-coated dishes than controls. Time course adhesion studies over 9 days showed that the heavy metal-induced fibrosarcoma cells progressively became more adherent to collagen type IV and less adherent to laminin- or fibronectin-coated dishes than did uninduced cells. However, with the removal of heavy metal from the medium, the HT-1080 fibrosarcoma cells were restored to their original adhesive potential. By phase microscopy, uninduced and induced HT-1080 cells demonstrated different morphological features and remained viable in an anchorage-dependent fashion on collagen type IV as a substrate. By way of contrast, GFAP-induced HT-1080 cells were not particularly viable in monolayer culture and readily detached from laminin as a substrate. The expression of beta1 integrin in GFAP-positive fibrosarcoma cells was decreased following heavy metal induction by Western blot analyses. In contrast, the expression of alpha2 integrin was increased whereas alpha5 integrin was unchanged in HT-1080 cells following the induction of GFAP. Gelatin zymography showed that 72 kDa collagenase was less expressed in GFAP-induced clones than in controls. Our data suggest that the forced expression of the intermediate filament, GFAP, in HT-1080 cells may modulate cell adhesion to different ECM substrates through alterations in expression of integrins.

Adenoviral vector-mediated gene transfer: timing of wild-type p53 gene expression in vivo and effect of tumor transduction on survival in a rat glioma brachytherapy model.

OBJECTIVE: This study sought to investigate modification of the radiation response in a rat 9L brain tumor model in vivo by the wild-type p53 gene (wtp53). Determination of the timing and dose of radiation therapy required the assessment of the duration of the effect of wtp53 expression on 9L tumors after in vivo transfection. METHODS: Anesthetized male F-344 rats each were stereotactically inoculated with 4 x 10(4) 9L gliosarcoma cells through a skull screw into the cerebrum in the right frontal region. Twelve-day-old tumors were inoculated through the screw with recombinant adenoviral vectors under isoflurane anaesthesia: control rats with Ad5/RSV/GL2 (carrying the luciferase gene), and study rats with Ad5CMV-p53 (carrying the wtp53 gene). Brain tumors removed at specific times after transfection were measured, homogenized, and lysed and wtp53 expression determined by Western blot analysis. Four groups of nine rats were, subsequently, implanted with iodine-125 seeds 15 days post-tumor inoculation to give a minimum tumor dose of 40 or 60 Gy. RESULTS: We demonstrated transfer of wtp53 into rat 9L tumors in vivo using the Ad5CMV-p53 vector. The expression of wtp53 was demonstrated to be maximum between days 1 and 3 post-vector inoculation. Tumors expressing wtp53 were smaller than controls transfected with Ad5/RSV/GL2 but this difference was not statistically significant. Radiation made a significant difference to the survival of tumor-bearing rats. Moreover, wtp53 expression conferred a significant additional survival advantage. CONCLUSION: The expression of wtp53 significantly improves the survival of irradiated tumor-bearing rats in our model.

Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression.

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

Astrocytoma cell interaction with elastin substrates: implications for astrocytoma invasive potential.

Elastin has been identified within the meninges and the microvasculature of the normal human brain. However, the role that elastin plays in either facilitating astrocytoma cell attachment to these structures or modulating astrocytoma invasion has not been previously characterized. We have recently shown that astrocytoma cell lines and specimens produce tropoelastin, and express the 67 kDa elastin binding protein (EBP). In the present report, we have established that astrocytoma cells attach to elastin as a substrate in vitro. The U87 MG astrocytoma cell line demonstrated the greatest degree of adhesion. In addition, all astrocytoma cell lines examined were capable of penetrating and migrating through an intact elastin membrane, and of degrading tritiated-elastin, a process that could be prevented by the pre-incubation of astrocytoma cells with EDTA, but not with alpha1-antitrypsin. Astrocytoma cells were also capable of penetrating 1 mm sections of human brain tissue maintained as organotypic cultures. Interestingly, the invasive potential of cultured astrocytoma cells plated on organotypic cultures of human brain was significantly increased after exposure to elastin degradation products (kappa-elastin), which interact with astrocytoma cell surface EBP. Our data show that astrocytoma cells express a functional 67 kDa EBP, enabling them to potentially recognize and attach to elastin as a substrate. These data also suggest that this elastin receptor may be involved in processes which regulate regional astrocytoma invasion.

The E2F-family proteins induce distinct cell cycle regulatory factors in p16-arrested, U343 astrocytoma cells.

We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, deltap34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2F's altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2F's uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.

Characterization of glial filament-cytoskeletal interactions in human astrocytomas: an immuno-ultrastructural analysis.

The role that glial filaments play in cells and tumors of glial origin is not well understood. We therefore undertook the present study to determine the relationships between glial and vimentin intermediate filaments (IFs), actin microfilaments, and CD44, a cell surface glycoprotein important in cell migration and invasion, in human astrocytoma cells. Three astrocytoma cell lines, U343 MG-A (U343), U251 MG (U251), and antisense GFAP-transfected U251 (asU251) were studied using immunofluorescence confocal and immunoelectron microscopy. Furthermore, we studied the phenotypic behaviour of these astrocytoma cell lines by analyzing their migration through Matrigel in vitro. U343 astrocytoma cells had the highest expression levels of glial fibrillary acidic protein (GFAP), whereas asU251 had virtually no expression of GFAP. Parental U251 cells had intermediate expression levels of GFAP. The elimination of GFAP expression in as U251 cells was accompanied by a marked increase in vimentin, actin microfilaments and CD44 levels. Gold labeling density counts of cytoskeletal and cell surface elements demonstrated that the differences between GFAP, actin, CD44 and vimentin levels in the different astrocytoma cell lines were statistically significant (p < 0.05). Results from the in vitro invasion assay revealed that U343 cells demonstrated the least invasive potential, whereas asU251 astrocytoma cells demonstrated the most. Our results show that elimination of GFAP expression by antisense leads to marked alterations in cell morphology and phenotypic behaviour. These data imply that GFAP may be linked spatially and functionally to cytoskeletal elements which may be altered when this IF is deleted in astrocytomas.

Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells.

Verotoxin 1 (VT1) is an E. coli toxin comprising an A subunit with N-glycanase activity, and five smaller B subunits capable of binding to the functional receptor globotriaosylceramide (Galalpha1-4-Galbeta1-4-Glcceramide-Gb3). VT is implicated in hemorrhagic colitis and the more serious hemolytic uremic syndrome. VT1 is active against various tumor cell lines in vitro and in vivo. To extend the anti-cancer spectrum of activity of VT to human brain tumors, in the present analysis we studied the effects of VT on the growth of 6 permanent human astrocytoma cell lines. All astrocytoma cell lines analyzed express Gb3 and were sensitive to VT-1 at a dose of 50 ng/ml, but sensitivity was not proportional to the relative Gb3 concentration. VT induced apoptosis in these cells was shown by electron microscopy. Morphological evidence (nuclear shrinkage and chromatin condensation) of apoptosis could be clearly distinguished 1.5 hrs after toxin addition. Ultrastructural preservation of organelles was observed in conjunction with blebbing of the plasma membrane, condensation of chromatin within the nucleus and nuclear shrinkage. Apoptosis was also induced by the recombinant toxin B subunit alone, suggesting that the ligation of Gb3 is the primary induction mechanism. These studies indicate that verotoxin/Gb3 targetting may provide a novel basis for the inhibition of astrocytoma tumour cell growth.

Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells.

We have characterized the expression and activity of the cell cycle regulatory machinery and the organization of the cytoskeleton of the p16(Ink4a)-deficient astrocytoma cell line, U343 MG-a (U343), following retinoic acid (RA) treatment. RA causes cell cycle arrest at low cell density and significant morphological changes in U343 cells, reflected by reorganization of the intermediate filament, GFAP, and actin. RA-induced cell cycle arrest is also associated with induction of p27Kip1 expression, inhibition of cdk2-associated kinase activity and alteration of the phosphorylation state of the pRB-family proteins. We next determined the effect of inducing expression of the cyclin dependent kinase inhibitors (CKI's), p16(Ink4a), p21Cip1/Waf1 or p27Kip1 on the proliferation and morphology of these malignant astrocytoma cells in the absence and presence of RA. Induction of p16, p21 or p27, using the tetracycline repressor system, potently inhibits proliferation of U343 cells. However, rather than resembling RA-treated cells, CKI-induced U343 cells become flat with abundant cytoplasm and perinuclear vacuolization. CKI-induced morphological alterations are accompanied by a significant reorganization of glial filaments within the cytoplasm. Interestingly, when U343 cells are growth arrested by p16, p21 or p27 induction and treated simultaneously with RA, a dramatic morphological change occurs, cells acquiring multiple long, tapering processes reminiscent of primary astrocytes. This rearrangement is accompanied by reorganization of GFAP, vimentin and actin. Vimentin specifically relocalizes to the tips of the long processes which form. The arrangement of intermediate filaments in these cells is, in fact, indistinguishable from their arrangement in primary human astrocytes. These data demonstrate that when a strong proliferative block, produced by CKI expression, occurs in conjunction with the morphogenic signals generated by RA, these p16-deficient malignant astrocytoma cells are induced to phenotypically resemble normal astrocytes.

Role of glial filaments in cells and tumors of glial origin: a review.

In the adult human brain, normal astrocytes constitute nearly 40% of the total central nervous system (CNS) cell population and may assume a star-shaped configuration resembling epithelial cells insofar as the astrocytes remain intimately associated, through their cytoplasmic extensions, with the basement membrane of the capillary endothelial cells and the basal lamina of the glial limitans externa. Although their exact function remains unknown, in the past, astrocytes were thought to subserve an important supportive role for neurons, providing a favorable ionic environment, modulating extracellular levels of neurotransmitters, and serving as spacers that organize neurons. In immunohistochemical preparations, normal, reactive, and neoplastic astrocytes may be positively identified and distinguished from other CNS cell types by the expression of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). Glial fibrillary acidic protein is a 50-kD intracytoplasmic filamentous protein that constitutes a portion of, and is specific for, the cytoskeleton of the astrocyte. This protein has proved to be the most specific marker for cells of astrocytic origin under normal and pathological conditions. Interestingly, with increasing astrocytic malignancy, there is progressive loss of GFAP production. As the human gene for GFAP has now been cloned and sequenced, this review begins with a summary of the molecular biology of GFAP including the proven utility of the GFAP promoter in targeting genes of interest to the CNS in transgenic animals. Based on the data provided the authors argue cogently for an expanded role of GFAP in complex cellular events such as cytoskeletal reorganization, maintenance of myelination, cell adhesion, and signaling pathways. As such, GFAP may not represent a mere mechanical integrator of cellular space, as has been previously thought. Rather, GFAP may provide docking sites for important kinases that recognize key cellular substrates that enable GFAP to form a dynamic continuum with microfilaments, integrin receptors, and the extracellular matrix.

Cyclin and cyclin-dependent kinase expression in human astrocytoma cell lines.

The expression of cyclins (A, B1, D1, D3, E), cyclin-dependent kinases (CDK2(3), CDK4), and the cyclin-dependent kinase inhibitors (CDKIs) p16(INK4A) and p21(CIP1) was studied in 9 malignant human astrocytoma cell lines using northern blot analysis, immunocytochemistry, and immunoblotting to see if their altered expression contributed to astrocytoma proliferation. Steady state cell cycle mRNA expression was analyzed in unsynchronized tumor cells, and cell cycle phase-specific gene expression was analyzed in 3 synchronized cell lines. Analysis of steady state expression revealed increased levels of several different cyclin transcripts and CDKs in a number of astrocytoma cell lines compared with normal human brain tissue or cultured fibroblasts. We confirm previous reports identifying loss of p16(INK4A) in astrocytomas, as a p16(INK4A) transcript was identified in only 2 cell lines and protein in 1 cell line. However, we now show that p21(CIP1) expression was also diminished relative to normal fibroblasts in all astrocytoma cell lines studied regardless of p53 mutation status. Analysis of synchronized astrocytoma cells revealed altered timing of mRNA expression of several cyclins. Immunocytochemistry revealed a generalized increase in immunoreactivity of astrocytoma cells for most cyclins and CDKs compared with human fibroblasts. Immunoblotting also revealed increased expression of cyclin proteins in a number of astrocytoma cell lines. These data suggest that increased expression of cyclins and CDKs, and decreased expression of CDKIs by human astrocytoma cell lines may contribute to their increased proliferative state. In addition, our data show that alterations in cell cycle genes in astrocytomas are not confined to the cyclin D1-CDK4-pRb axis.

Analysis of glial fibrillary acidic protein gene methylation in human malignant gliomas.

Glial fibrillary acidic protein (GFAP) is an intermediate filament specifically expressed in glial cells which helps to maintain and stabilize the glial cytoskeleton. Interestingly, with increasing astrocytic anaplasia, there is typically progressive loss of GFAP expression. In in vitro model systems, most permanent glioma cell lines are GFAP-negative. To determine the mechanism by which the transcription of the GFAP gene may be repressed in glioma cell lines, we initially performed a Southern analysis on a panel of human malignant glioma cell lines using a human cDNA probe for GFAP. By this method, no large rearrangements or deletions of the GFAP gene were found. Postulating that a change in methylation status of the GFAP gene could conceivably alter its expression in glioma cell lines, we studied the methylation state of the GFAP gene in the same glioma cell lines using a methyl-sensitive restriction enzyme digest of tumour and control DNA. Our analysis revealed that the GFAP gene was hypermethylated in 2/2 GFAP- negative glioma cell lines but not in 4/4 GFAP-positive glioma cell lines. To determine if methylation of CpG islands contained within the GFAP promoter could repress GFAP transcription, we designed deletional constructs from a 6 kb fragment of the mouse GFAP promoter, methylated them using Msp I- and Hpa II-methylases, and tested their activity in a standard CAT assay. Our data suggest that methylation of a 2 kb segment of the mouse GFAP promoter is sufficient to inactivate GFAP transcription. Our results further imply that methylation-mediated repression of GFAP transcription may be one candidate mechanism to account for decreased GFAP expression in certain human malignant glioma cell lines.

Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential.

Malignant astrocytomas are highly invasive tumors which infiltrate diffusely into regions of normal brain. The degradation of the extracellular matrix (ECM) by matrix metalloproteinases is thought to be one of the most important steps in the process of tumor invasion. However, the activity of most matrix metalloproteinases (MMPs) can be modulated by simultaneously secreted inhibitors (tissue inhibitors of metalloproteinases, TIMPs). We have previously shown that an imbalance between the levels of MMPs and TIMPs may be essential in the determination of the invasiveness of certain human malignant astrocytoma cell lines. To determine if the up-regulation of TIMP genes and gene products could modulate the invasiveness of human malignant astrocytoma cells, in the present study we have transfected a highly invasive astrocytoma cell line, SF-188, with an expression vector carrying a full-length TIMP-1 cDNA. The parental SF-188 astrocytoma cell line overexpresses the 72-kDa and 92-kDa type IV collagenases with little expression of TIMPs-1 and -2. Following transfection with TIMP-1, SF-188 astrocytoma clones expressed the 0.9 kb TIMP-1 message by northern analysis, and produced a 21 kDa metalloproteinase inhibitor by reverse zymography. The stable TIMP-1 SF-188 transformants demonstrated morphological changes and diminished growth rates in soft agar when compared to controls. The invasion of successfully TIMP-1 transfected astrocytoma cells across matrigel-coated filters was significantly decreased over controls. These results suggest that upregulation of TIMP-1 expression in SF-188 astrocytoma cells has decreased their in vitro invasive potential.

Characterization of a pineal region malignant rhabdoid tumor. Towards understanding brain tumor cell invasion.

The malignant rhabdoid tumor (MRT) of the central nervous system is a highly aggressive neoplasm of early infancy which is characterized by brain invasion and widespread dissemination along cerebrospinal fluid pathways. As the process of tumor invasion is mediated in part by the elaboration of proteases and protease inhibitors by tumor cells, we sought to determine the expression of the type-IV collagenases and their inhibitors (tissue inhibitors of metalloproteases, TIMPs) in an MRT from the pineal region of a 9-month-old male. In addition, as only a few reports exist concerning the cytogenetic abnormalities in MRTs, the cytogenetic features of this MRT were examined. When placed into tissue culture, the MRT grew vigorously in early passages. The cytogenetic analysis of the cells in passage one revealed a near diploid karyotype with some metaphases demonstrating monosomy 22. Northern analysis of type-IV collagenase transcripts revealed that the MRT expressed the highest levels of the 72- and 92-kD type-IV collagenase transcripts of any pediatric brain tumor examined. However, the MRT did not express any significant amounts of the TIMP-1 or TIMP-2 transcripts. By in situ hybridization analysis, the MRT demonstrated marked intratumoral expression of the type-IV collagenase but not TIMP transcripts. The results from this study suggest that this particular MRT may be a highly invasive brain tumor, at least in part on the basis of overexpression of the type-IV collagenases relative to the TIMPs.

Malignant rhabdoid tumour of the pineal region.

A 9-month-old male presented to hospital with signs and symptoms of raised intracranial pressure. A CT scan showed obstructive hydrocephalus from a large pineal region mass lesion into which an intratumoral hemorrhage had occurred. A posterior fossa craniectomy and subtotal excision of the mass lesion were performed. By histopathology, the lesion was a malignant rhabdoid tumour (MRT). Despite surgery and chemotherapy, the tumour grew inexorably, and the patient died four months after the initial diagnosis. MRT is a rare and highly invasive neoplasm which infrequently arises from the central nervous system. This is the first documented case of a MRT arising from the pineal region. The clinical, radiographic, and pathological features of the MRT in this patient are presented.

Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells.

Glial fibrillary acidic protein (GFAP)-positive astrocytoma cells were stably transfected with an expression vector carrying a murine complementary DNA for GFAP in the antisense orientation. Three stably transfected GFAP-negative transformants were identified by indirect immunofluorescence and expanded in vitro. The stably transfected and control cell clones were analyzed for morphological alterations, growth in monolayer and soft agar, adhesiveness, and in vitro invasive potential. In contrast to control astrocytoma cells which retained an astrocytic phenotype with polygonal or triangular cells extending multiple long and thin processes, the antisense GFAP-transfected cells demonstrated marked morphological alterations in the form of flat, epithelioid cells devoid of long, astrocytic glial processes. The antisense GFAP-transfected clones demonstrated a greater degree of cell crowding and piling at confluence than did controls. By tritiated thymidine analysis, the antisense GFAP-transfected cell clones demonstrated a 2-3-fold increase in incorporation of the radiolabel, suggesting an enhanced proliferative potential over controls. Antisense GFAP-transfected astrocytoma clones formed larger and more numerous colonies than did controls when tested for anchorage-independent growth in soft agar. Following a time-course adhesion assay, antisense GFAP-transfected astrocytoma clones were found to be less adherent to their substratum than controls. When assessed in an in vitro invasion assay system, antisense GFAP-transfected astrocytoma cells more readily penetrated Matrigel-coated filters than did controls. These data have shown that eliminating GFAP expression from astrocytoma cells has affected astrocytoma cell morphology and adhesion. The data also suggest that the growth and invasive potential of the antisense GFAP-transfected astrocytoma cells have been significantly enhanced by altering the expression of this glial-specific cytoskeletal protein in this experimental cell system.

Safety of anterior cement fixation in the cervical spine: in vivo study of dog spine.

There is increased use of methylmethacrylate in the treatment of neoplastic lesions of the spine. The advantage of this treatment is immediate stabilization, thus avoiding prolonged immobilization. In vivo study using a dog model showed that anterior fixation with cement, with or without Gelfoam as insulation material, produced no spinal cord injury.

Femoral head blood flow in long-term steroid therapy: study of rabbit model.

Using a rabbit model, previous studies showed steroid-induced hyperlipidemia with subsequent fatty embolization of the subchondral arteries and hypertrophy of the marrow fat cells, followed by elevation of femoral head pressure from the normal level of 25 cm to nearly 60 cm H2O after eight weeks of treatment. This has led us to believe that pressure changes lead to decreased blood flow in the femoral head. In our study of 22 New Zealand white adult rabbits, weighing an average of 4.0 kg, 14 received a weekly dose of 12.45 mg of methylprednisolone (Depo-Medrol), and eight served as control. Femoral head blood flow was established using the radioactive microsphere technique. Control and cortisone-treated rabbits had femoral head blood flow measured 6, 8 and 10 weeks after treatment. The average blood flow in the control femoral heads averaged 0.2039 +/- 0.076 ml/min/gm, with no difference in the left side and the right side. In the treated group, the average blood flow at ten weeks was 0.162 +/- 0.039 ml/min/gm on the right and 0.164 +/- 0.037 ml/min/gm on the left, which was significantly different. This is parallel to unpredictable clinical findings in human beings.

Comparative strengths of various anterior cement fixations of the cervical spine.

In vivo study, using cadaveric dog cervical spine, was performed in which the middle segment of the vertebral body was removed producing anterior instability. Eleven various methods of stabilization utilizing polymethylmethacrylate and other fixation devices were employed, and their static strength in hyperextension was tested and compared. A portion of the same dog's cervical spine in each case was used as a control. The results indicate that all fixation methods failed to regain the normal structural strength in extension. Cement with wire or chain methods of fixation were superior to other methods in this study. Fixation rigidity approaching rigidity of the normal spine appeared to be a significant factor determining the strength of the reconstructed cervical spine. Combined anterior and posterior fixation did not provide further strength, although it did increase the rigidity of the fixation.

Steroid-induced femoral head pressure changes and their response to lipid-clearing agents.

In rabbits, cortisone increases marrow fat cell size and intrafemoral head pressure, produces systemic fatty embolization, and probably is one of the causes of avascular necrosis of the femoral head. Clofibrate, a lipid-clearing agent, decreases the serum cholesterol level, reduces the marrow fat cell size changes, and decreases intrafemoral head pressure. This might be significant in improving femoral head venous outflow in long-term steroid-treated persons.

Cortisone-induced intrafemoral head pressure change and its response to a drilling decompression method.

Intramedullary head pressure changes were studied in a group of cortisone-treated New Zealand white rabbits. In addition to femoral head pressure measurements, serum cholesterol levels were followed serially and pathologic specimens from cortisone-treated femoral heads were obtained. Surgical decompression by a drilling method was performed and pressures were remeasured at four weeks following decompression. Persistent increases in cholesterol levels and in intrafemoral head pressures were noted, particularly from six to eight weeks after initiation of steroid treatment. Cholesterol levels were elevated to four times control values and femoral head pressures averaged 2 1/2 times the control values (60 cm of water vs 24.6 +/- 5.5 cm of water). Surgical decompression by drilling effectively reduced the once elevated pressures to control values. A relationship between increased fat cell size, increased intramedullary pressure and decreased femoral head blood flow in steroid-treated rabbits is postulated. The applicability of these data to humans with steroid-induced ischemic necrosis of the femoral head has yet to be demonstrated.