Computer-assisted stereology: point fraction of lung parenchyma and alveolar surface density in fetal and newborn sheep.

The aim of this study was to determine the accuracy of a new computer-assisted stereological technique in obtaining structural information of the lung. We compared the point fraction of lung parenchyma (Pp) and alveolar surface density (Sv) obtained by established manual point/intercept counting methods and compared them with those obtained using a computer-assisted method. Lung tissues obtained from normally grown fetal sheep (n = 6) and from newborn lambs with severe lung hypoplasia (n = 5) were inflation fixed via the trachea and processed for light microscopy. In verification-of-technique experiments, Pp and Sv correlated well with known values. There was a significant linear correlation between manual and computer-assisted stereological measurements for values of Pp (r2 = 0.92) and Sv (r2 = 0.98). Our data lead us to believe that the computer-assisted stereological technique described in this study provides accurate estimates of Pp and Sv and hence may be a valuable tool for evaluating the effects of factors upon structural development of the lung.

Multiple oblique illumination and high-definition microscopy for breast fine-needle aspirates.

The ability of multiple oblique illumination (MOI) and high-definition microscopy (Edge R-400 3-D microscope) to improve resolution of cellular detail in the evaluation of cytopathological specimens of Pap smears and thyroid fine-needle aspirates (FNAs) has been demonstrated. However, previous experiments showed that the advantages of MOI and high-definition stereo microscopy were less certain for the breast FNAs. We hypothesized that these findings were due to the lack of sample thickness for the breast FNA specimens. To test this hypothesis, we analyzed breast FNA specimens that were significantly thicker (10.5 microm). The number of lights (1, 2, 3, 4) and the angle of light (+1.5, 0, -3) were varied independently, creating 12 groups. Three images at each combination of settings were digitally captured and analyzed to obtain a histogram. The coefficient of resolution (Cr) was calculated to mathematically evaluate the grayscale histograms for intensities (0-255), where Cr = [¿IM - IN¿ x (N)] (IM, median pixel intensity; IN, measured pixel intensity; and N, number of pixels at given intensity). Mean Cr values demonstrated that the angle of light obliquity was not a factor in altering the resolution and contrast (p = .9). However, there was a significant increase in resolution, as measured by mean Cr values, as the number of lights was successively reduced from four lights to one light. Thus, the thicker specimen did show that increases in resolution were a significant function of the number of lights utilized.

Free fluid on abdominal computed tomography scan after blunt trauma does not mandate exploratory laparotomy in children.

BACKGROUND/PURPOSE: The isolated finding of free intraperitoneal fluid on abdominal computed tomography (CT) scan after blunt trauma in adults is considered an indication for laparotomy by many trauma surgeons. The authors wished to determine if these guidelines are applicable to children. METHODS: A retrospective chart review was conducted. The authors included all children (< or =12 years of age) sustaining blunt abdominal trauma who were admitted to our institution between January 1, 1994 and November 1, 1998. RESULTS: There were 814 children admitted, and 437 had abdominal CT scans. Thirty-four studies showed free fluid associated with solid organ injuries, spine or pelvic fractures, or pneumoperitoneum, and were excluded. Thirty-two children had free fluid without associated injuries and formed the basis for the study. Five of these children underwent laparotomy based on the CT finding alone. The remaining 27 were observed with serial abdominal examinations and did not require surgical intervention. Only 1 of the 5 children who underwent surgery for the finding of isolated free fluid had a therapeutic laparotomy. In comparison, during the same period, 38 children underwent laparotomy after blunt injury based only on physical examination findings with a therapeutic laparotomy rate of 68%. The therapeutic laparotomy rate was significantly higher when the procedure was based solely on clinical examination as compared with the isolated finding or free fluid on the abdominal CT (26 of 38 v 1 of 5, P < .05). CONCLUSION: In contrast to adults, finding isolated free fluid on abdominal CT scans in children after blunt trauma does not dictate immediate surgical exploration.

Vascularized bone marrow transplantation: A new surgical approach using isolated femoral bone/bone marrow.

BACKGROUND: Orthotopic composite tissue (limb) transplantation in rats is a unique model for vascularized bone marrow transplantation because bone marrow cells and bone marrow stroma are transplanted by microsurgical means, thus creating immediate bone marrow space and engraftment. However, it contains a skin component and other musculoskeletal tissues that complicate issues related to tolerance induction. MATERIALS AND METHODS: To study only aspects of vascularized bone marrow transplantation, we created a new isolated vascularized bone marrow transplant model in rats. The common iliac (or femoral) artery and vein were microsurgically anastomosed to the recipient abdominal aorta and inferior vena cava in an end-to-side fashion, respectively. Syngeneic male Lewis (RT1(1), n = 20) and allogeneic male BN (RT1(n), n = 10) donors were transplanted to female Lewis recipients. To establish rejection criteria, we examined histopathology and used the polymerase chain reaction (PCR) to assess microchimerism of donor male bone marrow cells in the peripheral blood of female recipients using rat Y chromosome (sex-determining region Y)-specific primers. RESULTS: All recipients were healthy and remained stable without major complications for up to 300 days posttransplant. Morphologically, syngeneic male Lewis bone marrow showed a near-normal appearance. Allogeneic male BN bone marrow was clearly rejected. Male bone marrow cells were detected by PCR in the peripheral blood of all syngeneic recipients, but not in allogeneic blood specimens. CONCLUSIONS: A new surgical approach to bone marrow transplantation was established. This consisted of the vascularized femoral bone/bone marrow transplant. Further analyses regarding the ability of vascularized femoral bone marrow transplants to induce systemic transplantation tolerance in adult rats will provide insights into not only various issues of immunology but also the potential clinical application of vascularized bone marrow transplantation.

Effects of microgravity on growing cultured skin constructs.

Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue. The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated. LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix. Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B). All groups were cultured for an average of 3 additional days. LSEs were processed for histologic evaluation. Skin cells were stained for cytokeratin to evaluate function. Images were digitally captured and processed for analysis. Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured. Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001). No significant difference in cellular function as measured by protein expression [stained cytokeratin area (micro(2)) per cell] was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227). In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups. Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001). Elimination of the air/liquid interface enhanced proliferation of keratinocytes. This effect was further enhanced in the presence of microgravity. No significant effect on cell function was noted with the use of this microgravity environment. We hypothesize that the increased epidermal contact plays a role in this proliferation. Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.

Use of three-dimensional microscopy with multiple oblique illumination in evaluating cytopathology preparations.

OBJECTIVE: To analyze whether the use of multiple oblique illumination (MOI), in contrast to axial illumination (AI), improves imagery in bright field microscopy. Specimens containing thick cell clusters, such as cervical Pap smears, are often misread because of out-of-focus cell clusters. We hypothesized that visualization of these specimens with MOI would enhance this information as compared with AI. STUDY DESIGN: Micrometer images and Pap smears were analyzed in focus, and 8 and 40 microns out of focus by MOI and AI. All fields were captured to a remote computer, histograms were constructed, and mean light intensity was calculated. Mathematical formulation was used to define the histogram distribution of the micrometer images. Statistical analysis was performed using one-way ANOVA and Newman-Keuls test. RESULTS: K(focus) was improved (P < .01) and at 20 and 40 microns out of focus, mean light intensities were greater (P < .003, P < .05, respectively) with MOI as compared to AI. CONCLUSION: MOI improves out-of-focus information by increasing light penetration through the specimen and enhancing contrast and resolution.

The effect of leukocyte infiltration on apoptosis in an in vitro thermal injury bioartificial living skin equivalent model.

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermally injured. The BSC was used as a target organ. Control BSCs without leukocytes (CON) were burned (BCON) by scalding with phosphate-buffered saline heated to 70 degrees C for 6 seconds, and they were then cooled with room temperature phosphate-buffered saline for 15 seconds. Human alloimmunocytes were added to CON to create rejection cultures (REJ) and to BCON to create burned rejection cultures (BREJ). Slides were stained with hematoxylin and eosin and anti-Lewis antibody. In situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Those sections that were immunostained for Lewis Y were analyzed for intensity stain index (ISI = [sigma P0 x I0]/ total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic cells / total tissue area. Necrosis and blister formation in the epidermal layer were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicators of apoptosis-were also present. Phagocytosis of keratinocytes by leukocytes was seen in REJ and BREJ. Immunostaining showed greater expression of Lewis Y antigen, as determined by ISI, in REJ as opposed to CON (58.2+/-2.3 vs. 36.4+/-2.3, respectively, P<.001), but no significant difference was found between BCON and BREJ (55.0+/-5.7 vs. 60.5+/-3.4, respectively) and REJ and BREJ (58.3+/-2.3 vs. 60.5+/-3.4, respectively). TUNEL staining indicated the presence of apoptosis as follows: REJ versus CON (0.0015+/-0.0002 vs. 0.0003+/-0.0001, respectively, P<.001); BREJ versus BCON (0.0031+/-0.0006 vs. 0.0018+/-0.0004, respectively, P<.05); REJ versus BREJ (0.0015+/-0.0002 vs. 0.0031+/-0.0006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The combination of these two variables results in increased apoptosis as determined by TUNEL. These findings suggest that a common pathway for skin injury may include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.

Pediatric trauma center criteria: an outcomes analysis.

BACKGROUND/PURPOSE: Trauma centers (TC) are certified based on widely accepted criteria. These specific criteria rarely are scrutinized individually. The purpose of this study was to analyze the individual components of a pediatric trauma center for their effect on outcome. METHODS: Members of the National Pediatric Trauma Registry were queried about the following: (1) separate pediatric emergency department (ED), (2) pediatric intensive care unit (PICU), (3) pediatric intensivist as PICU director, (4) pediatric surgeon as TC director, (5) in-house attending surgeon, (6) in-house pediatric emergency physician, (7) 24-hour operating room, (8) 24-hour computed tomography (CT) scan. Outcomes analyzed included mortality, length of stay, time in ED, days in PICU, and disability. Victims were stratified based on age (<7 or > or = 7 years) and severity of injury (ISS < or = 16, 17-35, > or = 36). Results were compared using Student's t test and chi2 analysis. RESULTS: A total of 59 of 74 centers responded, 18 were dropped because of low enrollment (mean, 1.6 patients). Questions 3, 4, 6, and 7 were eliminated because of skewed data. An in-house surgeon reduced the amount of time a mildly injured patient (ISS < or = 16) spent in the ED (210 v434 minutes), as did the separate pediatric ED (333 v592 minutes) and pediatric emergency physicians (344 v 507 minutes) in younger patients (> or = 7 years). An in-house surgeon reduced the morality rate in older (> or = 7) severely injured (ISS > or = 36) patients (46.7% v 56.8%; P < .05 for all). No other differences were significant. CONCLUSIONS: In-house personnel improved efficiency for the less severely injured, and an in-house attending surgeon reduced mortality in the severely injured older patient. None of the other variables were found to have a significant impact on outcome.

Fas-mediated induction of hepatocyte apoptosis in a neuroblastoma and hepatocyte coculture model.

BACKGROUND: We have previously demonstrated an increase in hepatocyte apoptosis when they are cocultured with neuroblastoma cells. Death receptors in the tumor necrosis factor (TNF) family such as TNFR1 and Fas have been identified as regulators of apoptosis and may be responsible for the altered regulation of apoptosis seen in our coculture model. To evaluate the effects of released factors and remove the potential alterations induced by direct contact, a noncontact coculture system was used to study the interaction between hepatocytes and neuroblastoma cells. METHODS: Human Chang hepatocytes (HC) were plated onto Falcon cell culture inserts with 0.45-micrometer pores in the permeable membrane. Human neuroblastoma cells (NB-IMR-32) were seeded into wells of the Falcon companion plate. After 24 h, inserts containing HC were placed into wells containing NB cells and incubated for 4 days. This provided a coculture environment without actual cellular contact. Immunohistochemical staining for TNFalpha, Fas, and Fas ligand (Fas-L) was performed. Apoptosis was detected via the TUNEL method. Images were analyzed with ImagePro-Plus. Statistical analyses were done with significance determined at P < 0.05. RESULTS: Chang hepatocytes demonstrated a significant increase in the levels of TNF, Fas, and Fas-L when cocultured with neuroblastoma cells (P < 0.005). In addition, the cocultured hepatocytes had a 20-fold increase in the apoptotic rate (P < 0.001). Neuroblastoma cells had no demonstrable level of Fas or TNF when grown alone and in cocultures. Neuroblastoma cells that were grown alone had an elevated level of Fas-L, but this level diminished by 44% when cocultured with hepatocytes (P < 0.001). CONCLUSION: An upregulated TNF/Fas receptor-ligand system may be responsible for increased apoptosis in hepatocytes when cocultured with neuroblastoma. This upregulation may be due to release of neuroblastoma-derived Fas ligand into the media. Tumors may alter the regulation of apoptosis in surrounding tissues via the death receptors.

Induction of apoptosis in a neuroblastoma and hepatocyte coculture model.

BACKGROUND: The dysregulation of apoptosis may alter the progression of tumor growth and explain the clinical dichotomy observed in children with neuroblastoma (NB). An overexpression of the bcl-2 proto-oncogene induces resistance to apoptosis and has been observed in unfavorable NB. We hypothesized that alterations in apoptosis may be a result of the interactions between NB and the tissues surrounding it. MATERIALS AND METHODS: Human Chang liver cells (HCL, 10(4) cells/cm2) were plated in two-chamber slides for 3 days. Human NB cells (10(5) cells/cm2) were added to one of the chambers and incubated for 3 more days. Control NB were plated under identical conditions in its own medium and in the HCL medium with growth curves measured. DNA fragmentation was detected via the TUNEL method (TdT-mediated nick end-labeling) and bcl-2 expression was determined by immunostaining. RESULTS: NB growth was unaltered by the change in medium. NB stained mildly positive for bcl-2 when plated alone but became markedly positive in coculture. Histologically, HCL and NB appeared healthy when plated alone, but a halo of apoptotic HCL was seen around NB in the coculture. When plated alone, both NB and HCL demonstrated minimal apoptotic activity as detected via the TUNEL method. In the coculture, a halo of HCL surrounding the NB exhibited markedly increased DNA fragmentation and this intensity diminished in cells distant from the NB. CONCLUSIONS: The regulation of apoptosis was altered in this coculture model of NB and HCL. HCL stimulated NB to overexpress bcl-2 and presumably become resistant to apoptosis. Conversely, NB induced the surrounding HCL to undergo apoptosis. The interaction between the local tissue and NB induced alterations in apoptosis in both cell types and resulted in a survival advantage for NB.

A novel in vitro model for xenorejection and immune mechanisms using bioengineered living skin equivalents.

We hypothesized that an in vitro bioengineered skin (LSE) could be used for the study of xenogeneic inflammatory or immunosuppressive mechanisms. Murine fibroblasts (10(4)/mL) were mixed with type 1 rat-tail collagen to form a matrix (approximately 5 days) on which human keratinocytes (NHEK, 10(5)/75 microL) were seeded. The xeno-LSE was used as a rejection target organ in vitro. Rejection was achieved by the addition of human lymphocytes (10(6)/75 microL). At various intervals of growth, control LSEs without lymphocytes (CON) and rejecting LSEs with immunocytes (REJ) were analyzed. Slides were stained with hematoxylin & eosin and examined under light microscopy. REJ xenocomposite LSEs showed classic histologic signs of rejection. There was separation of the D-E junction with the presence of dyskeratotic and necrotic cells with a concomitant inflammatory infiltrate. The CON LSEs showed essentially normal dermis (collagen fibroblast matrix) and keratinocytes. A significant finding was the separation of the D-E junction in the absence of an inflammatory infiltrate. Previous studies have shown that human keratinocytes can be grown with human fibroblasts that are histologically similar to natural human skin. This discohesive nature of the murine fibroblasts and human keratinocytes may represent an important intercellular interaction associated with xeno-cellular interactions and is worthy of further study. Furthermore, the ability to grow xeno-composite tissues has profound implications in the face of organ donor shortages. They may represent an eventual in vitro replacement of skin and even solid organs.

Pharmacologic inhibition of collagen in an experimental model of subglottic stenosis.

Subglottic stenosis occurs as a complication of prolonged endotracheal intubation secondary to inflammation with development of scar tissue and subsequent fibrosis. Collagen I and III levels increase during the healing process. Steroids alter the inflammatory response, decreasing recruitment of macrophages and fibroblasts. Beta-aminopropionitrile (betaAPN) inhibits the development of collagen cross-linking. A mechanism that would minimize hypertrophic scarring was sought. Eighteen dogs were anesthetized, had tracheostomies performed, and later had cautery of the mucosa and inner layer of the cricoid cartilage. Of 18 survivors, 6 animals were used as controls, 6 animals received oral Decadron, 2 mg/d, and 6 animals received oral betaAPN, 40 mg/d. There were 9 early deaths--5 in the steroid group. Animals were painlessly sacrificed, and the specimens were sectioned at the cricoid cartilage level and were stained immunohistochemically for antibodies to collagen types I to VI. Analysis of the area of scar and the intensity of stain was performed with Mocha image analysis software. Collagen III increased in control animals to 14.38 +/- 1.85 (intensity stain index), but this reaction was reduced by betaAPN (5.77 +/- 1.78, p < .01). Steroids had no significant effect on formation of any type of collagen. Lathyrogens (betaAPN) may offer a pharmacologic tool to reduce scar tissue.

In vitro bioartificial skin culture model of tissue rejection and inflammatory/immune mechanisms.

We hypothesized that an in vitro bioartificial skin rejection model using living LSEs grown in tissue culture could be developed for the study of autologous, allogenic, and/or xenogeneic inflammatory/immune mechanisms and topical immunosuppressive drugs. Human fibroblasts were mixed with type 1 rat-tail collagen to form a matrix (4 to 5 days), on which human keratinocytes were seeded. After a keratinocyte monolayer formed, CT cultures were raised to the air-liquid interface for continued growth. In the REJ LSE model, immunocytes isolated from human blood were seeded on top of the NHEK monolayer at the time of air-lifting. Thickness measurements of the acellular keratin and keratinocyte layers, and nuclear/cytoplasmic ratios, in both CT and REJ were made using digital image analysis. Immunostaining with anticytokeratin demonstrated a viable, keratin-producing epidermal layer; staining with anti-TGF-beta suggested a role for this cytokine in the rejection or wound-healing process. The LSE appeared histologically similar to normal human epidermis. Immunocytes added to the REJ cultures caused an obvious rejection response and were clearly identifiable in the gels as CD45+ staining cells. The LSE model appears promising for the study of immune/inflammatory mechanisms, thermal injury, screening antirejection agents that might be applied topically and as an in vitro replacement for skin graft studies in animals.

Rat renal allograft tolerance is associated with local TGF-beta and absence of IL-2r expression within chimeric immunocytic foci.

Tolerance was induced in Lewis (LEW) rat renal allograft recipients of Brown Norway kidneys by multiple pretransplant donor-blood transfusions and prior limited cyclosporine A. Rat renal allograft tolerance was associated with the induction of systemic donor T cells (10%), an early phase of nonspecific suppressor-cell generation, followed by maturation of systemic antigen-specific suppressor cells, and renal cellular infiltrates that develop long-term in situ in the kidney graft model. It was hypothesized that these infiltrates represent chimeric immunocytic foci that are locally regulated via a TGF-beta-dependent mechanism. Both immunohistochemical staining and digital image analysis for cellular and extracellular TGF-beta, IL-2 receptor (CD25), and the BN Class I-MHC marker (OX-27) were performed. Control rejecting (REJ) kidneys did not demonstrate any differences with respect to levels of infiltrating immunocyte area vs long-term surviving (TOL) kidneys (3.9% vs 4.5%, P = .303). Immunostaining with the BN Class I MHC marker (OX-27) demonstrated high levels of chimerism within immunocyte foci of the tolerant grafts (OX-27 BN+immunocytes 49.0% +/- 5.1%). In situ cellular IL-2 receptor (CD25) expression was demonstrated in REJ kidney infiltrates but not within TOL immunocytic infiltrating foci, when measured as percent of total lymphocytes (REJ = 5.0% vs TOL = 0.4%, P = .031). Conversely, TGF-beta expression was significantly higher in immunocytes of TOL kidneys when measured as the number of DAB chromogen-staining pixels per total immunocyte area (TOL = .076 vs REJ = .047, P = .003). In conclusion, these results suggested that stable mixed immune chimerism (SMIC) plays an important role in DST-CyA-induced tolerance in situ. SMIC-induced tolerance may involve a local TGF-beta-dependent mechanism that is associated with in situ TGF-beta (+) and IL-2r (-) immunocytes.

The effect of cyclosporine in gut ischemic injury: a computerized morphometric and enzymatic analysis.

PURPOSE: Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A (CSA), a potent immunosuppressant, attenuates immune/inflammatory cellular reactions. CSA also might be useful for inhibiting cellular immune responses involved in tissue ischemia/reperfusion injury. The authors hypothesized that CSA would attenuate inflammatory cellular changes associated with gut ischemic injury and that these effects could be quantified by computerized morphometry. METHODS: Twenty Sprague-Dawley rats underwent 60 minutes of gut ischemia by vascular occlusion of the superior mesenteric vessels. After 1 hour of reperfusion, the ischemic small bowel was harvested for histopathological examination and computerized morphometry, as well as xanthine oxidase (XO, U/mg protein) and maltase (MALT, mmol/L substrate degraded/min/mg protein) assays. CSA (5 mg/kg/d subcutaneously) was given to experimental animals (CSA, n = 10) for 5 days before ischemia, and vehicle was given to controls (CON, n = 10). The computer morphometric parameters studied were: surface index (SI, mucosal surface length per linear unit of intestine), average villous thickness (AVT), and average villous height (AVH). RESULTS: Results are provided in Table 1. CONCLUSION: The results of this study show that CSA may play a role in attenuating ischemia/reperfusion injury in the gut. Enzymatic analysis showed a beneficial role in the preservation of mucosal cell function after gut ischemia/reperfusion injury, as demonstrated by an elevated maltose level. Computerized morphometry demonstrated significant differences in all parameters in the experimental group, showing that CSA does confer gut mucosal protection during ischemia.

Airway measurement using morphometric analysis.

Measurement of airway size using endoscopic morphometry was investigated. Endoscopic images of the porcine tracheobronchial tree were obtained via a Storz system at a known distance from the telescope. The bronchi were marked with transluminal wires, transected, and imaged. All images were analyzed morphometrically with a computer image at multiple sites (N = 20). The endoscopic measurements were plotted against the macro lens images so that a straight line with a slope of 1 would indicate a consistent correlation. Area measurements for all images had a slope of 0.67 (r = .885); however, for images with area < 80 mm2 the slope was 1 (r = .879), for images with area < 80 mm2 of 0.93 (r = .875), and for images with area > 80 mm2 of -0.34 (r = .482), and major axis had a slope for all images of 0.73 (r = .904), for images with area < 80 mm2 of 0.88 (r = .923), and for images with area > 80 mm2 of -0.59 (r = .771). Accurate area measurements as well as major and minor axis measurements can be obtained for airways with area < 80 mm2 by means of this system. Each bronchoscope-telescope unit has an optimal target size for which measurements are accurate.

Graft-versus-host disease in extremity transplantation: digital image analysis of bone marrow in situ.

The development of either unstable immune chimerism and lethal graft-versus-host disease or stable immune chimerism and alloimmune tolerance can result from extremity transplantation. LBN rats served as recipients of Lewis vascularized extremity (limb) transplants. Recipients received no immune suppression and were immunologically unmodified. The bone marrow of transplanted and contralateral limbs was analyzed in situ for distribution of nuclei, nuclear area, and staining intensity by digital image analysis and computerized morphometry. Cellularity was significantly increased, and fat content was significantly decreased in the graft-versus-host disease animals' marrow versus the tolerant animals' marrow for both the transplanted and contralateral limbs. Tolerant animals demonstrated significantly increased nuclear staining compared with graft-versus-host disease animals for both transplanted and contralateral limbs. Additionally, there were significant changes between the host and the transplanted limbs for marrow intensity and cellularity within tolerant and graft-versus-host disease groups. The significant differences in the graft-versus-host disease-positive recipients suggested that both autoimmune dysregulation and alloimmune reactions were in effect for both donor and host bone marrow compartments. Cellular alterations in the tolerant recipients' marrow were suggestive of subtle subclinical graft-versus-host responses.

Morphometry and histochemistry of pulmonary arteries in a hypoplastic lung model.

Persistent pulmonary hypertension (PPH) is a common consequence of many neonatal respiratory diseases. The pathophysiology of PPH remains unknown. To study PPH, a rat model of pulmonary hypoplasia was used. Lung mass and body mass were recorded and lungs were prepared for frozen section examination and stained with hematoxylin and eosin, elastin, anti-Factor VIII, and nitro blue tetrazolium to identify nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The lungs were analyzed for air space volume, pulmonary artery wall thickness, total pulmonary arterial cross-sectional area, and density of tissue NADPH diaphorase staining. The mass of hypoplastic lungs was less than that of normal lungs (mean mass 85.93 mg vs 142.97 mg, P < 0.0001). The measured fraction of airspace volume was significantly less in hypoplastic lungs compared to controls (17.7% vs 30.8%, P < 0.0001). There was a significant difference in the pulmonary artery wall thickness ratio between the two groups (control 0.46 vs hypoplastic 0.487, P = 0.001). The arterial cross-sectional area was identical (control 1.25% vs hypoplastic 1.37%, P = 0.47). Staining density for NADPH diaphorase activity was determined using an intensity staining index (ISI). The experimental group showed increased staining for NADPH diaphorase (ISI = 54 in hypoplastic lungs vs 38 in controls, P < 0.01). Lung mass, appearance, and measured volume of airspace and tissue were all consistent with hypoplasia. In this model, arterial wall thickness was measurably greater in the hypoplastic group, while arterial cross-sectional area was not different. Staining for NADPH diaphorase showed significantly greater levels of enzyme in the hypoplastic lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer analyzed histology of ischemic injury to the gut.

Histopathologic alterations in the intestinal mucosa after ischemic injury have been extensively described in the literature, but these descriptions have primarily been qualitative in nature. The purpose of this study was twofold: (1) to establish parameters obtained by computerized digital image analysis that would be useful in identifying ischemic injury, and (2) to use these parameters to identify the critical period of intestinal ischemia producing measurable histopathologic change. Seventy male Sprague-Dawley rats weighing 80 to 150 g underwent various times of gut ischemic injury by vascular occlusion of the superior mesenteric artery and vein with a microaneurysm clamp. The clamp times were 0, 1, 20, 30, 40, 50, 60, 70, 80, and 90 minutes. Histological sections of the terminal ileum were quantitatively analyzed using Jandel Scientific's computerized morphometric image analysis system. Parameters studied were surface index (SI, surface length per linear unit of mucosa), average villous thickness (AVT), average villous height (AVH), and the number of villous cells/100 microns length (VC). Ischemic times of 1, 20, 30, and 40 minutes produced no measurable injury as compared with baseline (P[40 minutes versus baseline] = SI, .60; AVT, .84; AVH, .93; VC, .09). At 50 minutes, SI and AVH showed a measurable change from baseline (P[50 minutes versus baseline] = SI, .01; AVH, .02). Sixty minutes of ischemic time produced measurable change in all parameters (P[60 minutes versus baseline] = SI, .007; AVT, .001; AVH, .002; VC, .007).(ABSTRACT TRUNCATED AT 250 WORDS)