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Background/purpose: The effects of chemical and physical interactions in the microenvironment of solid tumors have not been fully elucidated. We hypothesized that acidosis, hypoxia, and elevated interstitial fluid pressure (eIFP) have additive effects on tumor cell biology and lead to more aggressive behavior during tumor progression. We investigated this phenomenon using three human osteosarcoma (OS) cell lines and a novel in vitro cell culture apparatus., Materials and Methods: U2OS, SaOS, and MG63 cell lines were cultured in media adjusted to various pH levels, oxygen tension (hypoxia 2% O2, normoxia 20% O2), and hydrostatic gage pressure (0 or 50 mmHg). Growth rate, apoptosis, cell cycle parameters, and expression of mRNA for proteins associated with invasiveness and tumor microenvironment (CA IX, VEGF-A, HIF-1A, MMP-9, and TIMP-2) were analyzed. Levels of CA IX, HIF-1α, and MMP-9 were measured using immunofluorescence. The effect of pH on invasiveness was evaluated in a Matrigel chamber assay., Results: Within the acidic-hypoxic-pressurized conditions that simulate the microenvironment at a tumor's center, invasive genes were upregulated, but the cell cycle was downregulated. The combined influence of acidosis, hypoxia, and IFP promoted invasiveness and angiogenesis to a greater extent than did pH, pO2, or eIFP individually. Significant cell death after brief exposure to acidic conditions occurred in each cell line during acclimation to acidic media, while prolonged exposure to acidic media resulted in reduced cell death. Furthermore, 48-h exposure to acidic conditions promoted tumor invasiveness in the Matrigel assay., Conclusion: Our findings demonstrate that tumor microenvironmental parameters - particularly pH, pO2, and eIFP - additively influence tumor proliferation, invasion, metabolism, and viability to enhance cell survival and must be controlled in OS research.

Purpose: We have previously shown that osteosarcomas have states of increased interstitial fluid pressure (IFP) which correlate with increased proliferation and chemosensitivity. In this study, we hypothesized that constitutively raised IFP in osteosarcomas regulates angiogenesis., Materials and Methods: Sixteen patients with the clinical diagnosis of osteosarcomas underwent blood fl ow and IFP readings by the wick-in-needle method at the time and location of open biopsy. Vascularity was determined by capillary density in the biopsy specimens. We performed digital image analysis of immunohistochemical staining for CD31, VEGF-A, VEGF-C and TPA on paraffin-embedded tissue blocks of the biopsy samples. Clinical results were validated in a pressurised cell culture system., Results: IFPs in the tumours (mean 33.5 +/- SD 17.2 mmHg) were significantly higher (P = 0.00001) than that in normal tissue (2.9 +/- 5.7 mmHg). Pressure readings were significantly higher in low vascularity tumours compared to high vascularity tumours (P <0.001). In the osteosarcoma cell lines, growth in a pressurised environment was associated with VEGF-A downregulation, VEGF-C upregulation and TPA upregulation. The reverse was seen in the OB cell lines. Growth in the HUVEC cell line was not significantly inhibited in a pressurised environment. Immunohistochemical assessment for VEGF-A (P = 0.01), VEGF-C (P = 0.008) and TPA (P = 0.0001) translation were consistent with the findings on PCR., Conclusion: Our data suggest that some molecules in angiogenesis are regulated by changes in IFP.

We have previously shown that osteosarcomas (OS) have states of increased interstitial fluid pressure (IFP), which correlate with increased proliferation and chemosensitivity. In this study, we hypothesized that constitutively raised IFP in OS regulates angiogenesis. Sixteen patients with the clinical diagnosis of OS underwent blood flow and IFP readings by the wick-in-needle method at the time and location of open biopsy. Vascularity was determined by capillary density in the biopsy specimens. We performed digital image analysis of immunohistochemical staining for CD31, VEGF-A, VEGF-C, and TPA on paraffin-embedded tissue blocks of the biopsy samples. Clinical results were validated in a pressurized cell culture system. Interstitial fluid pressures in the tumors (mean 33.5 +/- SD 17.2 mmHg) were significantly higher (p = 0.00001) than that in normal tissue (2.9 +/- 5.7 mmHg). Pressure readings were significantly higher in low vascularity tumors compared to high vascularity tumors (p < 0.001). In the OS cell lines, growth in a pressurized environment was associated with VEGF-A downregulation, VEGF-C upregulation, and TPA upregulation. The reverse was seen in the OB cell line. Growth in the HUVEC cell line was not significantly inhibited in a pressurized environment. Immunohistochemical assessment for VEGF-A (p = 0.01), VEGF-C (p = 0.008), and TPA (p = 0.0001) translation were consistent with the findings on PCR. Our data suggests that some molecules in angiogenesis are regulated by changes in IFP., ((c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Large structural allografts used for reconstruction of bone defects after revision arthroplasty and tumor resection fracture up to 27% of the time from osteolytic resorption around the fixation screw holes and tendon or ligament attachment sites. Treating structural allografts before implantation with bisphosphonates may inhibit local osteoclastic processes and prevent bone resorption and the development of stress risers, thereby reducing the long-term fracture rate. Taking advantage of allografts' open-pore structure, we asked whether passive soaking or positive-pressure pumping was a more efficient technique for delivering bisphosphonates. We treated matched pairs of ovine tibial allografts with fluids containing Tc-99m pamidronate and toluidine blue stain to facilitate indicator distribution analysis via microSPECT-microCT imaging and light microscopy, respectively. Surfactants octylphenoxy polyethoxy ethanol or beractant were added to the treatment fluids to reduce flow resistance of solutions pumped through the allografts. Indicator distribution after 1 hour of soaking produced a thin ring around periosteal and endosteal surfaces, while pumping for 10 minutes produced a more even distribution throughout the allograft. Flow resistance was reduced with octylphenoxy polyethoxy ethanol but unaffected with beractant. Pumped allografts displayed a more homogeneous indicator distribution in less time than soaking while surfactants enhanced fluid movement.

Background: The artificial lymphatic system (ALS), a mechanical system designed to reduce increased interstitial fluid pressure in solid tumors and enhance the delivery of chemotherapy, was evaluated within a randomized clinical trial treating spontaneously occurring canine appendicular osteosarcoma (OS), a tumor similar to its human OS counterpart., Methods: An ALS was investigated for its ability to increase OS blood flow and increase uptake of intravenously administered carboplatin., Results: Blood flow increased by 314% in tumors with active ALS drains versus 126% in control tumors (P < .03). Tumor carboplatin uptake increased by 51% after drain activation (P = .07). Microvascular density (MVD) was measured in tumors after surgical amputation and in corresponding bone regions in a cohort of normal dogs. The OS tumors had equivalent MVD as normal bone, and MVD was higher in the humerus than the femur (P < .03) in both tumor and normal bone. Median survival between the ALS-treated and control cohorts was not different despite increased drug uptake or ALS manipulation. Compared with historic controls, ALS drain insertion into tumors to reduce interstitial fluid pressure did not worsen the prognosis., Conclusions: The findings in canine spontaneously occurring OS indicate that an ALS may be of value as a chemotherapy adjunct for enhancing the delivery of chemotherapy to tumor interstitium.

A device is presented that positions ultrahigh molecular weight polyethylene (UHMWPE) debris against periprosthetic bone surfaces. This can facilitate the study of aseptic loosening associated with cemented joint prostheses by speeding the appearance of this debris within the periprosthetic space. The device, composed of a 100 microm thick bioabsorbable membrane impregnated with 1.4 x 10(9) sub-micron particles of UHMWPE debris, is positioned on the endosteum of the bone prior to the insertion of the cemented orthopedic implant. An in vitro pullout study and an in vivo canine pilot study were performed to investigate its potential to accelerate "time to aseptic loosening" of cemented prosthetic joints. Pullout studies characterized the influence of the membrane on initial implant fixation. The tensile stresses (mean+/-std.dev.) required to withdraw a prosthesis cemented into canine femurs with and without the membrane were 1.15+/-0.3 and 1.54+/-0.01 MPa, respectively; these findings were not significantly different (p > 0.4). The in vivo pilot study, involving five dogs, was performed to evaluate the efficacy of the debris to accelerate loosening in a canine cemented hip arthroplasty. Aseptic loosening and lameness occurred within 12 months, quicker than the 30 months reported in a retrospective clinical review of canine hip arthroplasty.

Elevated interstitial fluid pressure (IFP) is observed in most solid tumors. However, the study of the cellular processes of tumors and the development of chemotherapy are routinely studied using in vitro culture systems at atmospheric pressure. Using a new pressurized cell culture system, we investigated the influence of hydrostatic pressure on population dynamics of three primary osteosarcoma (HOS, U2OS, SaOS2) and two metastatic tumor cell lines (MCF7 breast, H1299 lung) that invade bone. Values of IFP in normal human bone and muscle, and in osteosarcoma tumors obtained during their surgical biopsy established the hydrostatic pressure range for the in vitro cell studies. The IFP values were obtained from a retrospective review of patient records. IFP from confirmed osteosarcoma was 35.9+/- 16.2 mmHg. Tumor IFP was significantly higher than muscle IFP (p < 0.001) and bone IFP (p < 0.003). The in vitro study measured the cell-line proliferation using hydrostatic pressures of 0, 20, 50 and 100 mmHg. The findings suggest that hydrostatic pressure either increases or decreases tumor proliferation rates depending on cell type. Furthermore, cell death was not associated with apoptosis.

Purpose: This study investigates the effect of constitutively raised interstitial fluid pressure on osteosarcoma physiology and chemosensitivity., Experimental Design: We did pressure and blood flow assessments at the time of open biopsy in patients with the diagnosis of high-grade osteosarcoma and correlated this to survival and chemotherapy-associated tumor necrosis. Osteosarcoma cell lines were then evaluated for proliferative and therapeutic indices in a replicated high-pressure environment., Results: Sixteen osteosarcomas in vivo were assessed and exhibited elevated interstitial fluid pressures (mean 35.2 +/- SD, 18.6 mmHg). This was not associated with significantly impeded blood flow as measured by a Doppler probe at a single site (P < 0.12). Nonetheless, greater chemotherapy-associated necrosis and associated longer survival were seen in tumors with higher interstitial fluid pressures (P < 0.05). In vitro, cells undergo significant physiologic changes under pressure. Osteosarcoma cell lines grown in a novel hydrostatically pressurized system had variable cell line-specific growth proportional to the level of pressure. They were more proliferative as indicated by cell cycle analysis with more cells in S phase after 48 hours of pressurization (P < 0.01). There was a significant elevation in the cell cycle-related transcription factors E2F-1 (P < 0.03) and E2F-4 (P < 0.002). These changes were associated with increased chemosensitivity. Cells tested under pressure showed an increased sensitivity to cisplatin (P < 0.00006) and doxorubicin (P < 0.03) reminiscent of the increased chemotherapy-associated necrosis seen in tumors with higher interstitial fluid pressure in the clinical study., Conclusions: The results of this study suggest that cells in the in vivo pressurized environment are at a higher state of regenerative activity than is demonstrable in conventional cell culture systems. Variations in tumor interstitial fluid pressure have the potential to alter chemotherapeutic effects.

Patellar tendon thickening (PTT) and patellar tendinosis (PTS) have been discussed in the veterinary literature as a post-operative complication of tibial plateau leveling osteotomy (TPLO). The purpose of this study was to define radiographic PTT, determine the frequency of and risk factors for PTT and PTS, and describe the clinical and histopathological findings of PTS after TPLO. We hypothesized that the location of the osteotomy alters forces placed on the patelloar tendon resulting in PTT or PTS. Radiographs and medical records from 83 dogs undergoing 94 TPLO procedures were retrospectively evaluated. Two months post-operatively, 19 dogs (20.2%) had a normal patellar tendon or mild PTT, 51 (54.3%) had moderate PTT, and 24 (25.5%) had severe PTT. Seven of the 24 dogs (7.4%) with severe PTT had clinical signs consistent with PTS. Only dogs with severe PTT developed PTS (p < 0.0001). The risk factors for the development of PTT include: a cranial osteotomy, a partially intact cranial cruciate ligament (CCL) in conjunction with a cranial osteotomy, and post-operative tibial tuberosity fracture. The only risk factor identified for the development of PTS was a partially intact CCL. Four dogs with PTS improved with conservative therapy and one improved with surgical treatment. Two dogs had tendon biopsies with histopothological review that showed tendon degeneration with lack of inflammation. As only the dogs with severe PTT develop PTS, a caudal osteotomy for the prevention of PTT and subsequent PTS is recommended.

Study Design: Three reconstruction options were evaluated biomechanically following total spondylectomy using human cadaveric spine specimens. OBJECTIVES.: To evaluate and compare the stability of combined anterior and posterior fixation incorporating poly-methyl-methacrylate with alternative accepted reconstruction techniques., Summary of Background Data: Total spondylectomy represents the most radical option for decompression in metastatic spinal cord compression. Poly-methyl-methacrylate is considered a useful adjunct in spinal column stabilization and arthrodesis; however, there is little published biomechanical data to support its use in this setting., Methods: Ten fresh-frozen human cadaveric spines (T9-L3) were used. After intact analysis, a total spondylectomy was performed at T12. Three potential reconstruction techniques were tested for their ability to restore stiffness to the specimen: 1) multilevel posterior pedicle screw instrumentation from T10-L2; 2) anterior instrumentation (ATL Z plate II) and rib graft at T11-L1 with multilevel posterior pedicle screw instrumentation from T10-L2; and 3) anterior cement (Simplex P) and pins construct (T12) with multilevel posterior pedicle screw instrumentation from T10-L2. Each of the three potential reconstruction techniques was tested on each specimen in random order using nondestructive testing under load control., Results: Only combined stabilization techniques (e.g., anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation and anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation) restored stiffness to a level equivalent to or higher than that of the intact spine in all loading modes (P < 0.05). Anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation provided more stability to the specimen than anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation in compression and flexion testing (P < 0.05). Posterior instrumentation alone did not restore stiffness to the intact level in compression and flexion testing (P < 0.005)., Conclusions: Combined anterior and posterior reconstruction using a cement construct provides equal to or more stability than the intact spine in all testing modes. Posterior stabilization alone is an inferior method of reconstruction following total spondylectomy. Poly-methyl-methacrylate has the advantage over traditional anterior reconstruction techniques in that it can be inserted using a posterior approach.

A well-distributed, patent microvascular network is essential for adequate, uniform delivery of chemotherapy into solid tumors. This network has not been evaluated in osteogenic sarcoma. Spälteholz tissue clarification was used to observe the microvasculature of canine humeri bearing osteogenic sarcoma. Freshly amputated limbs, obtained from therapeutic amputation, were infused with a micron-sized carbon particle solution, frozen, and then cut into sagittal and axial 0.5-mm thick sections. They were photographed, then radiographed using high resolution Faxitron xray, chemically treated to clarify the tissue, and then rephotographed. Microvasculature was identified by the localization of carbon particles, which were unaffected by the clarification process, within the clarified sections. Clarified section photographs were digitized to gray scale levels and analyzed using IMAGE software; levels are directly related to capillary density. Faxitron and original images were registered to the clarified images to identify tissue regions. Multiple regions of interest from normal muscle, fat, bone, and tumor regions were selected and averaged. The microvasculature of the tumor was inhomogeneous, whereas its density was considerably lower than normal adjacent muscle and bone (range, 56-72% lower). These findings suggest that insufficient microvascular density and distribution may provide additional explanation for the poor response of solid tumors to chemotherapy and radiation therapy.

Antineoplastic and antiresorptive drugs added to polymethylmethacrylate cement may prevent local cancer progression and failure of reconstructive devices used to treat patients with pathologic fractures. We tested the mechanical properties of cement containing various amounts of the drugs and found that as much as 2 g of either doxorubicin or pamidronate can be added to Simplex cement and the cement retains 87% of its compressive and tensile strength after 6 months of wet storage. Approximately 1 mg pamidronate elutes from experimental pellets. One half of the drug elution occurs within the first day in experiments that combined doxorubicin and pamidronate, and within 3 days when pamidronate was the only additive. Cement containing these drugs seems to be strong enough, but its fatigue strength should be tested before using it clinically. Sufficient amounts of the tested drugs elute to have potential biologic activity.

This study aims to characterize interstitial fluid pressure and blood flow in naturally occurring appendicular bone tumors in dogs because high pressure may influence the response of tumors to chemotherapy and radiation therapy. Eighteen client-owned dogs with naturally occurring appendicular bone tumors were included in this study. At the time of surgical biopsy, interstitial fluid pressure and blood flow were measured using wick-in-needle probes and laser Doppler flowmetry, respectively, within the soft tissue and bony components of the lesions and in normal muscle. Interstitial fluid pressure within the bony and soft tissue components of the tumors was significantly higher than interstitial fluid pressure in normal muscle. Blood flow in the bony component of the tumors was significantly lower than blood flow in normal muscle. There was no significant difference between blood flow in the soft tissue component of the tumors compared with that in normal muscle. Appendicular bone tumors in dogs have significantly higher interstitial fluid pressure and lower blood flow than do adjacent, unaffected soft tissues. The higher interstitial fluid pressure and lower blood flow may reduce tissue oxygenation and impede drug delivery. The effects of increased interstitial fluid pressure and decreased blood flow should be considered in the formulation of treatment strategies for the clinical management of appendicular bone tumors.

A mechanical drainage system, the "artificial lymphatic system" (ALS), consisting of a vacuum source and drain, is evaluated for its ability to aspirate the interstitial fluids responsible for the elevated interstitial fluid pressure (IFP) observed in solid tumors. IFP, pH, and pO2 radial profiles were measured before and after aspiration using wick-in-needle (WIN) probes, needle pH and oxygen electrodes, respectively. Laser Doppler flowmetry measured temporal changes in blood flow rate (BFR) at the tumor surface during aspiration. The WIN probe and IFP profile data were analyzed using numerical simulation and distributed mathematical models, respectively. The model parameter, P(E), reflecting central tumor IFP, was reduced from 15.3 to 5.7 mm Hg in neuroblastoma and from 13.3 to 12.1 mm Hg in Walker 256, respectively, following aspiration. The simulation demonstrated that spatial averaging inherent in WIN measurements reduced the calculated magnitude of the model parameter changes. IFP was significantly lower (p<0.05), especially in regions surrounding the drain, and BFR was significantly higher (p<0.05) following 25 and 45 min of aspiration, respectively; pH and pO2 profiles increased following aspiration. The experimental and mathematical findings suggest that ALS aspiration may be a viable way of reducing IFP and increasing BFR, pO2, and pH and should enhance solid tumor chemo and radiation therapy.

This paper presents findings from uptake studies to evaluate the ability of an "artificial lymphatic system" (ALS) to enhance large and small molecular weight drug transport into solid tumors and the therapeutic effect of the additional drug on their growth. These studies also served to test the effectiveness of an implantable multidrain ALS. Walker 256, Neuroblastoma, and Sarcoma dual-tumor models were used to evaluate the effect of ALS aspiration on the uptake of 3F8 monoclonal antibody, and doxorubicin. A tumor shrinkage experiment using Walker 256 dual tumors was used to evaluate the efficacy of an implantable ALS with cyclophosphamide chemotherapy. Drug uptake significantly increased in all aspirated tumors; 3F8 uptake was enhanced 37.4% in the Walker and 93.1% in the Neuroblastoma tumor lines (p<0.05). Doxorubicin uptake increased 23.2% in Sarcoma tumor (p<0.05). The shrinkage study demonstrated that one-drain aspirated tumors shrank 90% faster (p<0.01) than control tumors, while three-drain aspirated tumors shrank 123% faster than control tumors (p<0.01).

The present study was designed (1) to determine whether a free jejunal transfer in a large animal model can develop collateral circulation that is adequate to maintain viability after division of the pedicle and (2) to determine the earliest time pedicle ligation is safe after transplantation. A 15-cm jejunal segment was transferred to the necks of 18 dogs weighing 25 to 35 kg. The bowel segment was inset longitudinally under the skin on one side of the neck, partially covered by the neck muscles, and the mesenteric vessels were anastomosed to recipient vessels in the neck. The proximal and distal bowel stomas were exteriorized through skin openings 12 cm apart and matured. The dogs were subjected to ligation of the vascular pedicle at different intervals: postoperative day 7 (group I, n = 3), day 14 (group II, n = 5), day 21 (group III, n = 5), and day 28 (group IV, n = 5). Blood perfusion was measured in the proximal and distal bowel stomas before pedicle division (control) and 24 hours later using hydrogen gas clearance and fluorescein dye. Bowel necrosis was analyzed using planimetry. The bowel was also stained with hematoxylin and eosin and factor VIII, and new blood vessels were counted. Mean values (+/- standard deviation) were compared with control values for each test and with normal values in the intact bowel using analysis of variance with Neumann-Keuls post-hoc test for multiple comparisons. No jejunal free flaps survived when the vascular pedicle was divided 1 week postoperatively. Bowel survival was 60 percent at 2 weeks, 83 percent at 3 weeks, and 100 percent at 4 weeks. Hydrogen gas clearance values (ml/min/100 g) were 49.6 +/- 8.7 in the mucosa of the intraabdominal jejunum and 37.9 +/- 9.4 in the jejunum that was transferred to the neck before division of the pedicle. Twenty-four hours after pedicle division, hydrogen gas clearance values were 2.8 +/- 6.4 in group I (p < 0.05), 22.4 +/- 12.4 in group II, 23.9 +/- 9.3 in group III, and 34.2 +/- 7.5 in group IV. FluoroScan readings in the transferred jejunum were 201 +/- 7.2 in the control group, 9.3 +/- 2.8 in group I (p < 0.05), 79.1 +/- 10.6 in group II, 66.2 +/- 7.3 in group III, and 164 +/- 11.9 in group IV. New vessel formation as identified by factor VIII staining correlated with increasing bowel perfusion and flap survival rate. Bowel neovascularization, perfusion, and survival increased progressively 1 week after transfer. Significant portions of the transferred bowel will neovascularize and survive as early as 2 weeks postoperatively. However, a minimum of 4 weeks before ligation of the pedicle is necessary to maximize flap perfusion and guarantee survival.

Background: Changes in regional cerebral blood flow (rCBF) determined with H2(15)O positron emission tomographic imaging can identify neural circuits affected by centrally acting drugs., Methods: Fourteen volunteers received one of two midazolam infusions adjusted according to electroencephalographic response. Low or high midazolam effects were identified using post-hoc spectral analysis of the electroencephalographic response obtained during positron emission tomographic imaging based on the absence or presence of 14-Hz spindle activity. The absolute change in global CBF was calculated, and relative changes in rCBF were determined using statistical parametric mapping with localization to standard stereotactic coordinates., Results: The low-effect group received 7.5 +/- 1.7 mg midazolam (serum concentrations, 74 +/- 24 ng/ml), and the high-effect group received 9.7 +/- 1.3 mg midazolam (serum concentrations, 129 +/- 48 ng/ml). Midazolam decreased global CBF by 12% from 39.2 +/- 4.1 to 34.4 +/- 6.1 ml x 100 g(-1) x min(-1) (P < 0.02 at a partial pressure of carbon dioxide of 40 mmHg). The rCBF changes in the low-effect group were a subset of the high-effect group. Decreased rCBF (P < 0.001) occurred in the insula, the cingulate gyrus, multiple areas in the prefrontal cortex, the thalamus, and parietal and temporal association areas. Asymmetric changes occurred, particularly in the low-effect group, and were more significant in the left frontal cortex and thalamus and the right insula. Relative rCBF was increased in the occipital areas., Conclusion: Midazolam causes dose-related changes in rCBF in brain regions associated with the normal functioning of arousal, attention, and memory.

Experience with LDF has indicated that intraoperative estimation of cerebral blood flow can be very useful and that long-term recordings of cortical perfusion pressure are possible. The disadvantage of LDF is that measurements are extremely local and that the units of measurements are arbitrary. Moreover, the reliability of the technique depends to a large extent on probe position and on the avoidance of movement artifact. The disadvantages are, however, compensated by an excellent dynamic resolution with ultra-short time responses to sudden fluctuations in cerebral perfusion pressure. The high temporal resolution of LDF provides the opportunity to detect hemodynamic events and to monitor the microcirculatory effects of treatment that alters cerebral perfusion pressure in patients with raised intracranial pressure. At no point in the near future is LDF going to supercede continuous recording of intracranial pressure and mean arterial blood pressure and, hence, cerebral perfusion pressure. Rather, we envision LDF becoming an integral part of a multimodal patient monitoring system whereby time trends of monitored variables are combined with calculated variables to provide continuous assessment of cerebral hemodynamic and compensatory reserves.

The determination of small bowel perfusion after an ischemic insult is difficult. Regional perfusion was determined in an animal model of neonatal intestinal ischemia using the techniques of laser Doppler flowmetry and the clearance of locally generated hydrogen. Both methods reliably measured tissue perfusion in the areas of maximal ischemic injury. However, considerable variability, perhaps owing to motion artifact, was seen in areas of patchy necrosis. The results suggest that the laser Doppler flowmeter is a suitable technique to measure tissue perfusion in areas of maximal ischemia. However, efforts to reduce motion artifact will be necessary if the laser Doppler is to be used in tissue sites where blood flow is critical for safe anastomosis. This will be a subject of future study.

Background: We wished to determine whether transcutaneous oximetry or laser Doppler flowmetry (LDF) could identify patients at risk for wound failure after conservative, limb-sparing surgery for extremity sarcomas., Methods: Studies were performed on postoperative days (PODs) 1, 4/5, 7, and 9. Measurements of transcutaneous oxygen pressure (tcPO2) were taken at breathing room air (BL) and 100% oxygen (rate tcPO2). LDF measurements were taken at multiple sites along the wound, and a perfusion index was calculated., Results: Twenty-four patients were studied. Four (17%) had nonhealing wounds. There was no difference in tcPO2 (BL) values between healed and nonhealing wounds. Measurement of rate tcPO2 on POD 1 was significantly lower in the nonhealing wounds than in those with normal healing (28.5 +/- 12.1 mm Hg vs 14.3 +/- 16.2 mm Hg, mean +/- SD, p = 0.03). Rate tcPO2 values increased significantly in healing wounds from POD 1 to PODs 7 and 9 (p = 0.006, p = 0.009). This increase was absent in nonhealing wounds. A clear separation was noted in rate tcPO2 values between groups, with a minimum rate tcPO2 value recorded in a healed wound of 9 mm Hg/min, compared with the maximum value in a nonhealing wound of 7 mm Hg/min. The LDF perfusion index failed to predict wound healing at any of the measured time points., Conclusions: This study showed that measurement of tcPO2 during oxygen inhalation can accurately predict wound healing in patients after excision of an extremity sarcoma.

The growth, interstitial fluid pressure (IFP) and interstitial fluid velocity (IFV) profiles of a human neuroblastoma propagated in the flank of an immune suppressed rat were characterized. IFP was measured in the tumor center as a function of size, while radial distributions of IFP and IFV were measured in 2-cm tumors. IFP and IFV were measured using the wick-in-needle and clearance of locally generated hydrogen techniques, respectively. These techniques have a high spatial resolution, permit repetitive measurements, and are minimally invasive. We observed that IFP in the neuroblastoma increased as the tumor grew. Furthermore, IFP increased and its IFV decreased from the periphery toward the center of the tumor. Measured IFP and IFV values were compared to theoretical expectations calculated from the Baxter and Jain mathematical model. The predictions were highly correlated to the measured IFP and IFV profiles with the transport impedence parameter alpha 2 = 24.4. From our measurement data and the Baxter-Jain equations, we computed the interstitium hydraulic conductivity for neuroblastoma to be 7.11 x 10(-6) cm2/mm Hg-sec.

We present a method to assess quantitatively the immunological characteristics of tumours using radiolabelled monoclonal antibody and positron emission tomography (PET) to improve dosimetry for radioimmunotherapy. This method is illustrated with a glioma patient who was injected with 96.2 MBq of iodine-124 labelled 3F8, a murine antibody (IgG3) specific against the ganglioside GD2. Serial PET scans and plasma samples were taken over 11 days. A three-compartment model was used to estimate the plasma to tumour transfer constant (K1), the tumour to plasma transfer constant k2, the association and dissociation constants (k3, k4) of antibody binding, and the binding potential. Tumour radioactivity peaked at 18 h at 0.0045% ID/g. The kinetic parameters were estimated to be: K1 = 0.048 ml h-1 g-1, k2 = 0.16 h-1, k3 = 0.03 h-1, k4 = 0.015 h-1 and BP = 2.25. Based on these kinetic parameters, the amount of tumour-bound radiolabelled monoclonal antibody was calculated. This method permits estimates of both macrodosimetry and microdosimetry at the cellular level based on in vivo non-invasive measurement.

The potential usefulness of [C-11]-labeled alpha-aminoisobutyric acid (AIB) for tumor imaging has been demonstrated previously in our findings of increased tumor uptake with C-14-labeled AIB in human melanoma heterotransplants in nude mice, and subsequently, in a single case study using C-11 AIB to demonstrate the extent of metastases in patient with widespread malignant melanoma. We report here on the use of C-11 AIB in ten patients with metastatic or unresectable malignant melanoma. Five patients had intense tracer uptake at all known sites of tumor involvement. A sixth patient had good uptake in metastatic lesions in the shoulder and the pelvis, but did not demonstrate uptake within metastatic lesions in the lungs. Two patients had only minimal uptake over the tumor lesions while the two other patients had essentially normal studies. Further studies with C-11 AIB in patients with melanoma and other tumors are warranted.

In this paper we introduce and characterize pycnometry, a method used to measure fluid density, for determining a tissue's specific gravity. It uses a 2-ml glass pycnometer filled with distilled water to determine a tissue sample's displacement volume. The tissue's density is determined when it's weight is divided by this volume and specific gravity is computed by dividing the tissue density by the density of water. Pycnometry was validated using pre-calibrated glass, specific gravity standards over the range 1.03-1.26, and compared to the density gradient method using rat brain tissue. We observed that the specific gravity values obtained using pycnometry were highly correlated with the specific gravity standards (slope = 1.0107, r = 0.996) and with the density gradient column when tissue volumes larger than 0.120 ml were used with the pycnometer (slope = 1.0707, r = 0.9826). Good correlation was also observed between percent water content values computed using the Nelson equation with pycnometry or density gradient specific gravity values versus the measured percent water content values obtained with the wet weight/dry weight method. Pycnometry is an accurate, reproducible technique to measure tissue specific gravity and brain edema and is best suited for use in a laboratory that engages sporadically in brain edema measurement.

Cerebral blood flow response to changes in PaCO2 was studied in the edematous cerebral cortex of 19 patients with malignant supratentorial tumors using laser Doppler flowmetry technology. General anesthesia for craniotomy was induced with thiopental, 3-5 mg/kg i.v., and N2O, 60% in O2. In random sequence, 8 patients were assigned to receive fentanyl, 6 +/- 1.6 (SEM). mug/kg i.v.; the other 11 received isoflurane, 0.56% end-tidal + 0.07 (SEM). After a craniotomy bone flap was turned and the dura was opened, laser flowmetry probes were placed over surgically undisturbed cortex that was known to be edematous from preoperative CT and MRI scans. Flow index measurements were first made at hypocarbia (PaCO2 = 24.2 +/- 0.9 and 21.5 +/- 2.1 mm Hg for the fentanyl and isoflurane groups, respectively). Minute ventilation was then decreased and cortical flow index was remeasured with PaCO2 = 34.2 +/- 0.6 and 33.0 +/- 0.8 mm Hg for the fentanyl and isoflurane groups, respectively. Hypocarbia during fentanyl-supplemented N2O-O2 anesthesia resulted in a cortical flow index that was 70 +/- 8% of the flow index at near normocarbia (p <0.05). During isoflurane N2O-O2 anesthesia, however, there was a wide variety of responses to hypocarbia, including three patients whose flow indices increased markedly. The mean flow index during hypocarbia was significantly (p <0.05) lower during fentanyl-N2O anesthesia than it was during isoflurane-N2O anesthesia. There was no predictable relationship between the type of brain tumor and the CBF response to hypocapnia during isoflurane-N2O anesthesia. It is concluded that, in edematous brain, cerebral cortical blood flow response to hypocarbia is more likely to be preserved during fentanyl-supplemented N2O-O2 anesthesia than it is during isoflurane-supplemented N2O-O2 anesthesia. In neuropathologic states where hyperventilation is thought to be necessary to reduce cerebral blood flow and decrease brain bulk, isoflurane may be less satisfactory than fentanyl as a supplement to N2O-O2 anesthesia.

A novel method to measure specific gravity (SG) of tissues, pycnometry (PYC), is described. This method utilizes a 2ml glass pycnometer filled with distilled H2O to determine the displacement volume of a tissue sample and an equation to compute SG from the sample's weight and the pycnometer's weight before and after adding the sample. The PYC method was validated using glass SG standards over the range 1.02-1.26, and against the column density gradient (DG) method using brain tissue from 250-300 g male rats. Factors which affect PYC accuracy, i.e. sample size, were also evaluated. Our results indicate that PYC SG values are highly correlated with the glass SG standards (slope = 1.0107, r = 0.9955, p less than 0.001), and highly correlated with DG when approximately 0.120 ml tissue samples are used in the pycnometer. The DG method was preferable to the PYC method, however, when small tissue samples, i.e. 0.60 ml or less, were used.

To perform two independent regional blood flow measurements in tissue volumes of similar dimensions, we designed a hybrid blood flow probe capable of measuring regional perfusion by both laser-Doppler velocimetry (LDV) and H2 clearance. The probe consisted of two fiber-optic light guides to conduct light between the surface of tissue of interest and a laser-Doppler blood flowmeter. Also contained within the probe were a platinum 25-microns H2-sensing electrode and a 125-microns H2-generating electrode. The probe can thus be used to measure local perfusion with H2 clearance. The H2 can either be inhaled or can be generated electrochemically at the locus of interest. Evaluation of the probe in the canine gastric mucosa indicated 1) that the relationship between mucosal flow measurements made simultaneously with H2 clearance and LDV was highly significant and linear and 2) that H2 clearance could potentially be used to calibrate the laser-Doppler blood flowmeter in absolute units. The methods of constructing the flow probes are discussed in detail.

Quantitative radionuclide scintigraphy often requires empirical calibration factors derived from phantoms which simulate the radioactivity distribution, tissue geometry and tissue composition of the region of interest. This paper describes a method in which casts made with fiberglass tape of the region of interest. This paper describes a method in which casts made with fiberglass tape are used to form realistic, water-fillable phantoms of the limbs. Phantoms were constructed for the hind legs of the dog and rabbit, species frequently used in developing new radioscintigraphic techniques. Leg bones removed from euthanized animals were mounted anatomically within the casts. The dimensions of the phantom cavities were determined by x-ray computed tomography. A procedure was developed for orienting the phantoms to match the hind leg geometry of a given experimental setup. Use of the phantoms for image activity calibration is illustrated for a geometric-mean counting technique used to determine 99mTc activity densities in soft-tissue regions of the dog thigh. Generalization of the calibration technique to planar and tomographic imaging is straightforward. In situ measurements of 99mTc activity density obtained by external counting were compared with in vitro radioassays of excised tissue. For 22 tissue samples obtained from four dogs, the in situ and in vitro data were linearly correlated (r = 0.98, p much less than or equal to 0.001) over a 50-fold range of activity density. The mean and standard deviation of the observed percent discrepancies [% discrepancy = 100 (in situ - in vitro)/in vitro] were (7.8 +/- 2.9) and (13.7 +/- 2.1), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the feasibility of measuring gastric mucosal blood flow by laser-Doppler velocimetry (LDV), we utilized two LDV flowmeters to monitor blood flow in mucosa and serosa of chambered canine stomach. In isolated, nonautoregulating gastric segments vasodilated with isoproterenol, LDV mucosal and muscularis blood flows were both linearly related to total electromagnetic blood flow during step increases in perfusion pressure. To assess the depth of the LDV measurement, we recorded reactive hyperemia following arterial occlusion. Reactive hyperemia was frequently registered in the mucosa but rarely in muscularis. Placing a layer of nonperfused mucosa-submucosa between the probe and the perfused mucosa abolished the resting LDV mucosal flow signal and attenuated the recording of peak hyperemia by 85%. Furthermore, intra-arterial infusions of both adenosine and isoproterenol frequently increased LDV mucosal flow and decreased LDV muscularis flow, although total flow was consistently increased. These findings indicate that our LDV instruments yield linear, superficial measurements of gastric blood flow in either mucosa or muscularis. Although calibration in absolute units remains to be achieved, our results demonstrate that LDV is a practical means of studying the gastric mucosal microcirculation.

We are studying the transport of C-11 and N-13 labeled amino acids in tumor-bearing rabbits to determine the role of amino acid transport in the pathogenesis of muscle wasting in cancer. To validate a new, in vivo, method for measuring transport in skeletal muscle with these compounds, an isolated hindlimb model was developed in rabbits. The limb was perfused with a non-recirculating, normothermic, constant pressure system and a cell-free perfusate. Hemodynamic and metabolic parameters were measured during the first 75 min. of perfusion and found to remain normal and stable. Flow varied directly with perfusion pressure over the normal range of resting flows in the intact rabbit hindlimb. Time-activity curves (TAC's) were recorded from the medial thigh following bolus co-injection of L-[amide N-13] glutamine or N-13 L-glutamate with Tc-99m human serum albumin (HSA) into the femoral artery. Regional plasma flow was determined from the Tc-99m data. The N-13 TAC's consistently manifested a three-phased washout with half times of approximately 30 sec., 5 min. and 2 hr. Capillary and cellular transport parameters were computed from the N-13 data using a double barrier, single capillary model of capillary and cellular transport and assuming that the three washout components result, respectively, from tracer throughput, extraction into the interstitial space and extraction into the intracellular space. This interpretation was validated and the sensitivity of the technique to transport processes demonstrated by examining the effects on the N-13 TAC's and computed transport parameters of several factors known to influence cellular transport of amino acids, viz., the insulin concentration, amino acid concentration and pH of the perfusate. Time-activity curves and transport parameters for N-13 L-glutamine in the isolated limb were very similar to those observed in the intact rabbit hindlimb, suggesting that studies in the perfused model are indicative of amino acid transport in vivo. The methodology described here is especially well suited for studying the specific effects on transport of factors which influence amino acid metabolism in skeletal muscle (e.g., hormones and monokines).

A new technique, laser-Doppler flowmetry, has been used intraoperatively to measure blood flow responses in normal brain tissue and brain tumor to blood pressure and arterial blood gas alterations. We have observed that blood flow is reduced in most cerebral tumors, and that most tumors retain the normal response to changes in arterial blood gas; however, these responses are varied. One group of tumors in our study demonstrated an autoregulatory capacity; a second behaved passively--that is, blood flow changes followed blood pressure--while a third showed no response.