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Biomedical subjects

T M Krummel

Publications and source records attributed to T M Krummel.

At least 55 records · Page 3Linked to original sources
PubMed1997

Maintenance of fetal murine pulmonary microvasculature in heart-lung en bloc whole organ culture.

The authors have previously shown that murine fetal lungs can be maintained in serum-free whole organ culture and that airway ligation accelerates lung development. In spite of extensive use of lung organ culture systems, the vasculature of the unperfused lung in organ culture has not been studied. The aim of the present study was to compare organ cultures of heart-lung blocks with continuous perfusion of the pulmonary vasculature to those without perfusion, ie, whole lungs cultured without the attached heart. Time-dated pregnant CD-1 mice were killed on gestational day (Gd) 14. The fetuses were removed via laparotomy. Heart-lung blocks and whole lungs without the heart were excised under sterile conditions and cultured in BGJb media. Some of the heart-lungs and whole lungs underwent tracheal ligation whereas others were left with the trachea unligated allowing free egress of airway fluid. After 7 days, the cultured organs were processed for histology, ultrastructural analysis, and immunohistochemistry. (1) Lungs were fixed in 10% formalin, paraffin embedded, and processed for routine H&E staining. (2) Lungs were fixed in 2.5% glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy. (3) Lungs were embedded in CRYOform and flash frozen for immunohistochemical localization of PECAM-1 (CD31) (PECAM-1, Platelet endothelial cell adhesion molecule-1, a selective endothelial cell marker). Our daily observations of the cultured organs showed that the heart maintained synchronized beating for all 7 days in culture. Perfusion of the pulmonary microvasculature was demonstrated. Light microscopically, H&E sections showed that fresh fetal Gd14 pseudoglandular lungs (time-zero) had a defined capillary network, which was more centrally localized and peripherally less developed. The presence of more numerous lung capillaries in the cultured heart-lung blocks was noted when compared with cultured lungs alone. Ultrastructurally, endothelial cells with intact structural integrity were identified only in cultured whole lungs with hearts. Immunohistochemical staining of the whole lungs with rat antimurine PECAM-1 monoclonal antibody performed on cryosections showed the presence of vasculature by specific PECAM-1 localization on endothelial cells. PECAM-1 labeling of capillaries was noted in Gd14 (time-zero) lungs. In addition, the lungs cultured with hearts, ie, perfused lungs, showed more well defined, distinct capillary networks stained with PECAM-1 antibody than unperfused lungs without hearts. Our results showed that microvasculature is present in murine fetal lungs at Gd14. After 7 days in organ culture, the maintenance of lung microvasculature was confirmed histologically, ultrastructurally, and immunohistochemically. The microvasculature in whole lungs cultured as perfused/beating heart-lung blocks was better maintained than the microvasculature of unperfused whole lungs cultured without hearts. A perfused whole lung organ culture model is attractive because the lung architecture is better maintained and may be useful in lung developmental studies as it mimics the in situ heart-lung functional physiological relationship.

Animals↗
PubMed1997

Regenerative healing of incisional wounds in midgestational murine hearts in organ culture.

OBJECTIVE: Using an organ-culture fetal heart repair model, we explored fetal repair in tissues other than dermis. METHODS: Wounded fetal mouse hearts of 14 and 18 days' gestation (term = 20 days), as well as hearts of 22 days' gestation (newborn), were maintained in serum-free medium. Specimens were fixed at 2, 7, and 11 days and then processed for histologic examination. Small fragments of fetal hearts from all time points were cultured as explants. The migration of cells from the periphery of the explants was compared at day 4, and the pattern of microfilaments in these cells was assessed. RESULTS: In 14-day hearts (n = 18), tissue architecture was rapidly reestablished without an inflammatory response or scarring, constituting regenerative repair. In 18-day hearts (n = 18), no reestablishment of muscle fibers or wound closure occurred. In the 22-day explants (n = 12) the wounds closed by scarring. Cell migration from 14-day explants was 4.7 +/- 2.3 ocular units; from 18-day explants, it was 2.6 +/- 1.1 ocular units; and from 22-day explants, it was 0.9 +/- 0.4 ocular units. Microfilaments of 14-day cells were arranged at the periphery of the cell consistent with cardiomyocytes. Microfilaments of 18- and 22-day cells were arranged in parallel arrays (stress fibers) that were consistent with fibroblasts. CONCLUSIONS: We propose that regenerative healing of 14-day fetal hearts is by the migration of cardiomyocytes. At 18 and 22 days, cardiomyocytes are too differentiated and unable to migrate; hence cell migration is limited to resident fibroblasts, which are deficient at 18 days but sufficient at 21 days to be repaired by the scarring process.

Animals↗
PubMed1997

Nitrofen dose-dependent gestational day-specific murine lung hypoplasia and left-sided diaphragmatic hernia.

2,4-Dichlorophenyl-p-nitrophenyl ether (nitrofen) is known to induce pulmonary hypoplasia (PH) with or without diaphragmatic hernias (DH) in rats and mice. We determined the timing of administration and dose of nitrofen needed to create left-sided DH and PH in fetal mice. Time-dated pregnant CD-1 mice were gavaged with various doses of nitrofen in the later one-half of gestational days (GD) 8-11. Fetuses were removed by laparotomy at GD 14, fixed, and evaluated histologically. Fetal lung size was inversely related to nitrofen dose. Morphometric analysis of normal and nitrofen-exposed hypoplastic lungs at the pseudoglandular stage revealed significant differences in lung length, surface area, and in the number of airways. Left-sided DH were observed in a "dorsolateral" position accompanied by PH in approximately 30% of GD 14 fetuses exposed to 25 mg nitrofen on GD 8. A minimal portion of liver was present in the hernia. The lungs of fetuses exposed on GD 9, 10, and 11 progressed to near normal size. Murine fetuses exposed to 25 mg nitrofen on GD 8 resulted in PH and DH, whereas other doses created dose-dependent PH alone or none at all on GD 11. Our study established that, to create left-sided DH and PH in murine fetuses, nitrofen dose specificity and time of administration during gestation were crucial.

Animals↗
PubMed1996

Bronchial ligation enhances murine fetal lung development in whole-organ culture.

Evidence exists from both congenital anomalies and animal models that normal fetal lung development is dependent on maintenance of fluid pressure within the developing "airways." Fetal tracheostomy, allowing free egress of airway fluids, results in lung hypoplasia, indicating that some airway distending pressure is required for normal lung development to occur. In contrast, fetal tracheal ligation, which increases fetal airway pressure, reverses lung hypoplasia in animal models. The authors' experiments test the hypothesis that large airway obstruction accelerates the development of murine lungs in vitro in whole-organ culture. Fetuses from time-dated pregnant CD-1 mice at day 14 of gestation were removed (term, 20 days), and the lungs were excised. The left bronchus of each lung was ligated (n = 26), after which the left lung was isolated and cultured at 37 degrees C (95% air, 5% CO2) in BGJb media supplemented with vitamin C and antibiotics. Some fetal lungs were cultured under similar conditions without bronchial ligation (n = 11). After 7 days in culture, the lungs were taken for various analyses. The lungs were fixed in either formaldehyde and processed for paraffin embedding for light microscopic evaluation and morphometric data collection, or were freshly minced and aliquots taken for total protein and DNA content. Several more ligated and unligated lungs were processed for ultrastructural analysis. Morphometric analysis on transverse sections of lungs showed significant differences in the lung tissue size, thickness, epithelial cell height, luminal areas, perimeters, and total number of airspaces (airway + primordial alveolar airspaces). It was evident that bronchial ligation promoted lung development. The ligated lungs displayed thinning of the primordial alveolar walls with cuboidal epithelial cells. The total number of airspaces per field was lower for better developed ligated lungs because of the increased area of airspaces compared with that of the unligated lungs. The dorsoventral tissue thickness (in micrometers) of the ligated lungs was significantly greater than that of the unligated lungs (124.1 +/- 7.0 v 89.6 +/- 8.0); the average outer perimeter of the primordial alveolar airspaces was greater for ligated lungs (404.56 +/- 19.0 microns v 256.85 +/- 17.0 microns). Similarly, the luminal diameter of the spaces of ligated lungs was almost double that of the unligated lungs (38.0 +/- 2.0 microns v 20.3 +/- 2.0 microns), as was the luminal surface area. The morphometric data, which suggest enhanced maturation of the ligated lungs, are supported by results of ultrastructural studies. Ligated lungs had significantly more lamellar bodies. Although total protein and DNA content were greater among the ligated lungs, the protein/DNA ratios did not differ among the groups. The intraluminal pressure (airway pressure) of ligated lungs was 2.9 mm Hg and 3.1 mm Hg at 2 and 4 days in organ culture; the respective pressures for unligated lungs were 1.0 mm Hg and 0.8 mm Hg. These data support the hypothesis that mechanical distending pressure resulting from airway obstruction not only improves pulmonary architecture but also accelerates lung development in vitro. Although these effects have been seen in in vivo models, this is the first proposed in vitro organ culture model. This model may prove to be a powerful tool for the study of molecular mechanisms of mammalian lung development with respect to mechanical and chemical (cytokines, hormones) stimuli.

Airway Obstruction↗
PubMed1995

Regenerative healing of incisional wounds in murine fetal lungs maintained in organ culture.

Although fetal dermal repair is known to be fundamentally different from adult healing, the response to wounding in other organs is less well characterized. Scarless repair in mid-gestation dermis with a transition to adult-type healing at term has been shown in fetal organ culture. A lung explant culture system was used to investigate whether wound repair in the fetal lung shows characteristics similar to those found in fetal dermis. Lungs from 14-day and 18-day Cd-1 murine fetuses and 2-day-old newborns, (term = 20 days, n = 24) were wounded by linear incision and incubated at 37 degrees C, in a 21% O2, 5% CO2 environment, in BGJb supplemented with vitamin C and antibiotics. Medium was changed daily. Samples were fixed at 7 days and embedded in paraffin. Sections were stained with hematoxalyn-eosin and Masson Trichrome. Additional 14-day and 18-day samples were frozen in freon and immunohistochemical staining for TGF-beta performed. Other frozen tissues from each time point were homogenized and used to assay for endogenous TGF-beta levels by Western blot analysis. Histology showed reconstitution of tissue architecture across the wound in 14-day and 18-day specimens. In representative histological sections, intact bronchial architecture developed across the previous wound site. No cellular inflammatory response was observed, and collagen deposition was undetectable at the site of the wound by Trichrome staining. By 22 days the lung explants showed a much less ordered repair, including disorganized collagen deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Nitric oxide reversal of recurrent pulmonary hypertension and respiratory failure in an infant with CDH after successful ECMO therapy.

Nitric oxide (NO) represents a new therapeutic modality for treating neonatal pulmonary hypertension and may obviate the need for extracorporeal membrane oxygenation (ECMO) in a number of cases of neonatal respiratory failure. Recently, the authors treated an infant with a congenital diaphragmatic hernia and pulmonary hypertension with NO on two separate occassions. During the initial period of stabilization, NO failed to reverse the pulmonary hypertension and prevent the development of progressive respiratory failure. After a successful course of ECMO, recurrent pulmonary hypertension developed that was successfully treated with continuous low-dose NO therapy for over 1 month. Prolonged administration of NO in varying doses titrated to clinical and echocardiographic parameters was well tolerated by the infant and prevented the need for a second run of ECMO.

Extracorporeal Membrane Oxygenation↗
PubMed1995

Perforated appendicitis: past and future controversies.

Although mortality rates for pediatric appendicitis have been reduced to near zero with the development of safe surgical procedures and routine perioperative antibiotic therapy, the incidence of perforated appendicitis in children has remained at a comparatively high level. This article reviews the recent literature on perforated pediatric appendicitis, including a discussion of some of the controversies concerning management of the ruptured appendix. In addition, the future of both the evaluation and treatment of this disease, including laparoscopy, the possibilities for outpatient antibiotic therapy, and the worrisome potential for a change in referral patterns as children with this disease are increasingly enrolled in managed care plans, are considered.

Appendicitis↗
PubMed1994

The extracellular matrix of the fetal wound: hyaluronic acid controls lymphocyte adhesion.

Adhesive interactions between lymphocytes and components of the extracellular matrix (ECM) within a wound environment play a crucial role in determining the inflammatory response following tissue injury. In fetal wounds the extracellular matrix is composed predominantly of hyaluronic acid. Within this environment the inflammatory reaction as a result of injury is minimal. We propose that this lack of an inflammatory cell response in the fetal wound is due to the high levels of hyaluronic acid within the ECM and the inability of lymphocytes to adhere to this matrix component. Therefore, we examined the adhesive properties of fetal lymphocytes to fibronectin, vitronectin, collagen types I, III, IV, V, and hyaluronic acid--ECM components involved in fetal and adult wound environments. Fetal lymphocytes from both spleen and thymus demonstrated significant binding capabilities to fibronectin, vitronectin, and collagen types I and III. No intrinsic binding capabilities were detected to hyaluronic acid. Adhesion was not affected by the addition of IL-1, IFN-gamma, or phorbol dibutyrate. The inability of lymphocytes to adhere to hyaluronic acid helps to explain the lack of inflammation found in fetal wounds and serves to demonstrate the importance of ECM-lymphocyte interactions in determining the inflammatory response during fetal wound healing.

Animals↗
PubMed1994

Platelet-derived growth factor induces fetal wound fibrosis.

The fetal response to cutaneous injury differs markedly from that of the adult, proceeding with only minimal inflammation, minimal fibroblast proliferation, and only essential collagen deposition. Although the sequence of events in adult wound healing is well defined and thought to be controlled in part by potent polypeptide cytokines, relatively sparse information exists regarding growth factor involvement in fetal wound repair. Thus, the authors sought to examine the effect of platelet-derived growth factor (PDGF), a putative adult wound healing regulator, on the cellular and extracellular matrix events at a fetal wound site. SILASTIC wound implants containing 0, 1.0, 5.0, or 10.0 ng of human PDGF were placed subcutaneously on the backs of 24-day-gestation fetal rabbits (full term, 31 days) and then harvested after either 1, 3, or 5 days in utero. The specimens underwent standard histological processing and were evaluated in a blinded fashion. Compared with controls, PDGF-treated implants had a marked increase in acute inflammation, fibroblast recruitment, and collagen and hyaluronic acid deposition; these differences appeared to be largely time- and PDGF dose-dependent. Thus, the fetal system is responsive to an adult wound healing mediator, and these data suggest that fetal repair proceeds in the absence of PDGF.

Animals↗
PubMed1994

Endoscopic sphincterotomy in the management of posttraumatic biliary fistula.

Complications after nonoperative management of hepatic trauma are rare but include persistent biliary fistula in 4% of cases. Therapy usually involves surgical drainage or hepatic resection to control the fistula. The authors present a case of hepatic biliary fistula treated nonoperatively with percutaneous drainage and endoscopic sphincterotomy. A 16-year-old girl suffered a grade III parenchymal liver fracture to the right lobe in an automobile accident. She was hemodynamically stable with no coexistent injuries and was treated nonoperatively. Over 2 weeks her total bilirubin rose to 3.2 mg/dL, and alkaline phosphatase was 463 U/L. Ultrasound showed free intraperitoneal fluid, and 2 L of bilious fluid were retrieved by paracentesis. A radionuclide scan confirmed massive extravasation of isotope from the right lobe. Two drains percutaneously placed over the parenchymal fracture produced 500 to 600 mL of bile daily. Endoscopic retrograde cholangiopancreatography (ERCP) 1 week later showed a normal extrahepatic biliary system. A 1-cm sphincterotomy was performed without difficulty. Within 72 hours, percutaneous drains produced only 35 mL per day. The follow-up radionuclide scan showed no evidence of extrahepatic biliary extravasation, and 3 weeks later the drains were removed. Six months after the accident, results of the computerized tomography scan and liver function tests were normal. It was believed that endoscopic sphincterotomy reduced fistula output by decreasing the intrabiliary pressure caused by the ampulla, thus favoring internal drainage. This case demonstrates the effectiveness of endoscopic sphincterotomy as an alternative to direct surgical intervention for the management of posttraumatic biliary fistula.

Accidents, Traffic↗
PubMed1993

Hyaluronate uptake and CD-44 activity in fetal and adult fibroblasts.

One of the unique aspects of fetal wounds is the persistence of a high concentration of hyaluronic acid (HA) in the extracellular matrix. Within the wound environment, both the synthesis and breakdown of this complex glycosaminoglycan are regulated predominately by the fibroblast with cell binding mediated by the membrane receptor CD-44. The ability of adult and fetal fibroblasts to incorporate fluorescent-labeled HA (F-HA) was measured, and the effect of a monoclonal antibody to CD-44 to block this uptake was studied. Fetal fibroblasts incorporated significantly greater F-HA than adult fibroblasts at the 4- and 6-hr times (P < 0.05), while CD-44 blockade reduced the uptake to background levels. These results demonstrate that fetal fibroblasts incorporate F-HA more rapidly than adult fibroblasts and that this uptake is mediated by the membrane protein CD-44.

Aging↗
PubMed1993

Recombinant tissue plasminogen activator for neonatal and pediatric vascular thrombolytic therapy.

Thrombotic vascular occlusion may complicate the clinical course of many neonatal and pediatric pathologic processes. Systemic thrombolytic therapy with heparin, urokinase, or streptokinase may not be appropriate in the critically ill neonate because these agents generate a diffuse coagulopathic state. Direct surgical intervention for repair may be precluded by the small size of the vessels involved. Recombinant tissue plasminogen activator (rTPA) induces only a minimal proteolytic state while inducing thrombolysis within the local environment of the clot. We report our experience with regional rTPA infusion in four critically ill patients with venous and arterial thrombotic disorders. there were two brachial artery occlusive lesions--a neonate with iatrogenic occlusion due to a misplaced intravenous catheter and a 2-year-old child with inadvertent arterial ligation during an attempted venous cutdown. Two venous lesions consisted of a full-term neonate with renal vein/inferior vena caval thrombosis and a 32-week infant with partial superior vena caval thrombosis due to a Broviac catheter. Systemic thrombolytic therapy was contraindicated in these patients because of underlying illnesses. Pretherapy vascular evaluation included Doppler examination and angiography. The rTPA infusion was continued until there was evidence of clot lysis by ultrasound, angiogram, or venogram. Infusion rate of rTPA was adjusted according to fibrinogen levels. All three neonates responded successfully to rTPA therapy. Two neonates required only bolus administration and one responded to combined bolus and continuous infusion therapy after 58 hours. rTPA failed to reverse brachial artery occlusion in the 2-year-old child with purpura fulminans.(ABSTRACT TRUNCATED AT 250 WORDS)

Brachial Artery↗
PubMed1993

Isolated fetal mouse limbs: gestational effects on tissue repair in an unperfused system.

While it is known that adult tissue repair is tightly regulated through local effects in the wound environment mediated by circulating blood elements such as platelets, white cells, cytokines, and hormones, the degree to which each is required in fetal tissue repair is uncertain. This raises the following questions regarding regulation of fetal tissue repair: (1) is the local wound matrix alone sufficient to sustain fetal tissue repair in the same regenerative manner seen in previous in vivo whole animal studies? (2) Will it occur only during the period of rapid fetal growth and development in early and mid gestation? To address these fundamental questions, an organ culture system has been designed to grow isolated, unperfused, developing fetal mouse limbs in a chemically defined, serum-free media. Amputated fetal mouse forelimbs (n > or = 10) were wounded with linear incisions at gestational days 14, 16, and 18 (term = 19); the wounds were closed primarily. These amputated and wounded limbs were placed on steel grids in organ culture petri dishes, then partially submerged in a chemically defined, serum-free media. The limbs were grown at 37 degrees C in humidified 95% air/5% CO2 for 1 week. These wounded limbs were examined histologically at days 0, 3, and 7 postwounding to determine their viability and whether or not tissue repair occurred. In the 14-day group, limb growth and differentiation was evident during the incubation period. Normal dermal and epidermal architecture was restored at the wound site without abundant collagen deposition by day 7 postwounding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amputation, Traumatic↗
PubMed1993

The potential role of the lymphocyte in fetal wound healing.

Wound healing is a complex process involving a number of different cell populations. Since fetal wounds contain only a small number of mononuclear cells and no fibrosis immunohistochemical techniques using monoclonal antibodies specific to T and B lymphocytes, as well as macrophages, were used to identify the mononuclear cells in fetal wounds and to compare the lymphocyte response with the adult wound healing response. Polyvinyl alcohol sponges were placed subcutaneously in fetal rabbits on day 24 of gestation (then 31 days). Adult rabbits underwent similar implantation. Implants (8 to 14 per group) were harvested 5 days later and examined by H&E and trichrome staining as well as immunohistochemical staining with monoclonal antibody (Mab) specific for rabbit T and B lymphocytes. The sponges in one group of fetal rabbits were impregnated with the polypeptide growth factor, transforming growth factor-beta (TGF-beta), a known regulator of adult tissue repair. Adult wounds showed an intense inflammatory response with extensive collagen deposition; 80% of the infiltrating cellular elements were T lymphocytes. Fetal wounds were less cellular with minimal collagen deposition; 40% of the cells were T lymphocytes. The fetal wounds treated with TGF-beta were intensely cellular and fibrotic with 71% of the cellular infiltrate comprised of T lymphocytes. The increased cellularity of the TGF-beta wounds appears to be primarily the result of increased numbers of T lymphocytes. These findings demonstrate that the fetus mounts an attenuated T lymphocyte response compared to the adult. The increased response of T lymphocytes with TGF-beta suggests that the specific growth factor milieu may account for the fetal response to wounding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts.

Fetal wound healing is characterized by minimal inflammation, mild fibroplasia and rapid, but organized collagen deposition such that scarring is not apparent. The matrices of fetal wounds differ greatly from adult wounds in that fetal wounds are persistently enriched with hyaluronic acid (HA). It has been shown that a reduction in fetal rabbit wound HA results in an adult-like healing response with increased fibroplasia and neovascularization. These observations suggest that HA can modulate cellular activity in fetal repair. Therefore, this study was designed to define the effect of HA on fetal fibroblast function. Fibroblasts from the skin of fetal rabbits were isolated and maintained in culture medium containing either no HA (controls), 1 microgram/ml, 10 micrograms/ml or 100 micrograms/ml of HA (n = 6 for each group). Fibroblast proliferation was quantitated by DNA content in each culture, and collagen and noncollagen protein synthesis were analyzed by incorporation of [3H] proline into collagenase-digestible and collagenase-nondigestible protein, respectively. At all concentrations tested, HA significantly inhibited fetal fibroblast proliferation (p < 0.02), but stimulated collagen (p < 0.002) and noncollagen protein (p < 0.005) synthesis. These findings provide further evidence that HA affects the function of fetal fibroblasts. Moreover, this study in conjunction with previous in utero findings suggests that HA may have a regulatory influence in scarless fetal healing by affecting cellular function during the repair process.

Animals↗
PubMed1993

In vitro and in vivo analysis of the inability of fetal rabbit wounds to contract.

Fetal rabbit wounds that are sutured show excellent repair without obvious scarring. In contrast, an unsutured wound in a rabbit fetus does not close, and it appears that the process of wound contraction does not occur. Experiments were carried out to illustrate the mechanisms responsible for the noncontraction of open fetal rabbit wounds. Results showed that the lack of wound contraction was not an artifact caused by rapid fetal growth. With regard to the ability of cultured fetal fibroblasts to show cytoplasmic muscle-induced cell contraction, we found that, in cultured fetal fibroblasts, cell contraction was induced by adenosine triphosphate. Contractile abilities of fetal-derived fibroblasts were equivalent to those of adult-derived fibroblasts. The fetal fibroblasts also demonstrated the generation of superior contractile activity when examined in a fibroblast-populated collagen lattice model. Finally, the ability of amniotic fluid to alter wound contraction was addressed by means of the fibroblast-populated collagen lattice in vitro model. Increasing concentrations of amniotic fluid inhibited fetal fibroblast lattice contraction. Therefore, rabbit amniotic fluid contains an inhibitor that may be partially responsible for the noncontraction of fetal rabbit wounds in utero.

Journal Article↗