Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)-positive putative oligodendroglial progenitor cells.

AIMS: Limited remyelination is a key feature of demyelinating Theiler's murine encephalomyelitis (TME). It is hypothesized that a dysregulation of differentiation of oligodendroglial progenitor cells (OPCs) represents the main cause of insufficient regeneration in this model of multiple sclerosis. METHODS: TME virus (TMEV)-infected SJL/J mice were evaluated by footprint analysis, light and electron microscopy, immunohistology, confocal immunofluorescence and RT-qPCR at multiple time points ranging from 1 h to 196 days post infection (dpi). RESULTS: Footprint analysis revealed a significantly decreased stride length at 147 and 196 dpi. Demyelination progressively increased from 14 towards 196 dpi. A mild amount of remyelination was detected at 147 and 196 dpi. Early onset axonal injury was detected from 14 dpi on. TMEV RNA was detectable throughout the observation period and markedly increased between 7 and 28 dpi. Intralesional nerve/glial antigen 2 (NG2)-positive OPCs were temporarily increased between 28 and 98 dpi. Similarly, a transient upregulation of NG2 and platelet-derived growth factor alpha-receptor mRNA was noticed. In contrast, intralesional 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-positive oligodendrocytes were decreased between 56 and 196 dpi. Although CNPase mRNA remained unchanged, myelin basic protein mRNA and especially its exon 2 containing splice variants were decreased. Glial fibrillary acidic protein (GFAP)-positive astrocytes and GFAP mRNA were increased in the late phase of TME. A mildly increased colocalization of both NG2/CNPase and NG2/GFAP was revealed at 196 dpi. CONCLUSIONS: Summarized, the present results indicated a dysregulation of OPC maturation as the main cause for the delayed and limited remyelination in TME. A shift of OPC differentiation from oligodendroglial towards astrocytic differentiation is postulated.

Confocal laser scanning analysis of an equine oral mast cell tumor with atypical expression of tyrosine kinase receptor C-KIT.

A 20-year-old female horse showed a nodular, firm, focal ulcerated mast cell tumor at the right dorsobuccal face of the tongue. Histologically, the nonencapsulated tumor consisted of dense, infiltrating aggregates of well-differentiated, Cresyl violet-positive mast cells accompanied by numerous eosinophils. Furthermore, they exhibited a strong, diffuse, intracytoplasmatic immunohistochemical signal for tryptase and a faint membrane-associated and perinuclear signal for tyrosine kinase receptor KIT. Confocal laser scanning microscopy confirmed an aberrant spatial colocalization of KIT in the Golgi apparatus, which may be the result of a defective protein processing within the tumor cells. The tumor was not associated with a poor prognosis.

Effects of anti-glaucoma drugs timolol and GLC756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, on endotoxin-induced-uveitis and -arthritis in rats.

Anti-glaucoma drugs exhibiting anti-inflammatory properties are desirable for the long-term treatment of glaucoma since they may reduce the risk for treatment-related inflammatory processes in outer compartments of the eye. The purpose of this study was to evaluate potential anti-inflammatory effects of two topically and systemically applied anti-glaucoma drugs i.e. GLC 756, a novel mixed dopamine D2 receptor agonist and D1 receptor antagonist, and timolol a beta-adrenoceptor antagonist using endotoxin-induced uveitis (EIU) and arthritis in rats as an in vivo model. For EIU, 8-week-old Lewis rats were intravenously injected at 160 microg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone, as a positive control, were either topically (0.4%, 0.5%, and 0.1%, respectively, 16-times 20 microL eye drops during 48 h) or systemically (1mg/kg subcutaneous for 5 days) administered. Cell infiltration in tissue of the eye and knee joint were assessed histopathologically and in special compartments of the eye by confocal microscopy 48 h after LPS-induction. Numerous infiltrating cells were detected in the eyes after LPS-induction and half of the animals showed arthritis. Topical and systemic pre-treatment with GLC756 and timolol resulted in reduced cell infiltration in the eye. In addition, GLC756 reduced, whereas timolol increased the incidence of arthritis. Betamethasone suppressed almost completely the cell infiltration in the eye and the incidence of arthritis. In conclusion, the observations that GLC756 reduced cell infiltration in the eye and the incidence of arthritis after LPS-induction is compatible with anti-inflammatory properties of this drug. By contrast, timolol produced no consistent anti-inflammatory effect since both inhibitory as well as stimulatory effects on inflammatory processes were seen.

Immunohistochemical study about the Flt-1/VEGFR1 expression in the gastrointestinal tract of mouse, rat, dog, swine and monkey.

Fms-like tyrosine kinase 1 (Flt-1) performs a subordinate effector role in mesenchymal angiogenesis and potentially serves an equally important functional role as a self-contained receptor in epithelial cells. In both endothelial cells and epithelial cells, Flt-1/vascular endothelial growth factor receptor 1 (VEGFR1) downstream signalling is involved in regulating cellular processes such as cytoskeletal changes and cellular survival protection. Cellular renewal of the gastrointestinal mucosa is based on these processes and might involve Flt-1/VEGFR1 pathway activities; the molecular mechanisms regulating these cellular dynamics remain unclear. This study was performed to investigate the presence and distribution of Flt-1/VEGFR1 in epithelial cells of the gastrointestinal tract by immunohistochemistry (IHC). Gastrointestinal tissues were taken from eight anatomical sites from mouse, rat, dog, swine and monkey. Present results revealed a cytosolic Flt-1/VEGFR1 staining pattern in mucosal epithelial cells for all investigated species. Non-epithelial structures also displayed a distinct Flt-1/VEGFR1 positivity and included vascular smooth muscle walls, enteric smooth muscle layers, the enteric nervous system and capillary endothelial cells. Diverse intensities of the Flt-1/VEGFR1 binding reaction within each species were observed in the intestinal mucosa with a strong immunoreaction in enterocytes and with a low protein expression in the ileum in most species. Crypt cells in the large intestine were mostly negative for Flt-1/VEGFR1. A peculiar and mainly intranuclear antibody binding reaction was found in Brunner's gland epithelial cells of mouse and rat whereas Brunner's glands of dog, swine and monkey remained completely negative. These results indicate a potential involvement of Flt-1/VEGFR1 in normal restitution of gastrointestinal structures in the species studied. Additionally, intranuclear Flt-1/VEGFR1 antibody binding in Brunner's glands of rodents may suggest a nuclear translocation of the transmembrane VEGFR1 which has not previously been described.

Effects of C1 inhibitor and r-SP-C surfactant on oxygenation and histology in rats with lavage-induced acute lung injury.

OBJECTIVE: To assess the effects of C1 inhibitor (INH) administration and r-SP-C surfactant application on oxygenation and lung histology in an acute respiratory distress syndrome model. DESIGN AND SETTING: Randomized, controlled experimental study in an animal research laboratory. MATERIAL: 36 adult male Sprague-Dawley rats. INTERVENTIONS: Animals were subjected to repetitive lung lavage. Four experimental groups and two control groups were studied: groups 1 and 2 served as controls. Animals of groups 3-6 received 200 U/kg body weight C1-INH (group 3), 25 mg/kg r-SP-C surfactant (group 4) or both (group 5) at 60 min postlavage (pl). Animals of group 6 were treated with 200 U/kg C1-INH1 at 10 min pl. Animals of group 1 were killed 60 min (min) pl, animals of groups 2-6 were killed at 210 min pl. Thereafter the lungs were excised for histological examination. MEASUREMENTS AND RESULTS: Hyaline membrane formation, intra-alveolar neutrophil (PMN) accumulation and intra-alveolar/perivascular haemorrhage were graded semiquantitatively (0-4). Blood gases were determined 120, 150, 180 and 210 min pl. At 210 min pl pO(2) in group 4 (456+/-74 mmHg) and group 5 (387+/-155 mmHg) was significantly higher than in controls (72+/-29 mmHg) or after C1-INH monotherapy (group 3: 120+/-103, group 6: 63+/-12 mmHg). PMN infiltration after C1-INH monotherapy was significantly less severe than in controls. The combination of r-SP-C surfactant and C1-INH led to significantly lower PMN infiltration than surfactant monotherapy. CONCLUSION: In this lavage-induced acute respiratory distress syndrome model the administration of C1-INH might be followed by a higher clinical efficacy of exogenously supplied recombinant SP-C surfactant.

Additive effects of phosphodiesterase-4 inhibition on effects of rSP-C surfactant.

We have tested the effects of combined treatment with a recombinant surfactant protein C based surfactant (rSP-C surfactant) containing a phosphodiesterase-4 (PDE-4) inhibitor, roflumilast, in a lung lavage model of acute lung injury. The following groups were tested: (1) controls receiving sham exposure; (2) PDE-4 inhibitor (6.0 mg/kg body weight, intratracheally) alone; (3, 4) rSP-C surfactant (25 and 100 mg phospholipids [PL] per kg body weight) alone; and (5, 6 ) treatment with rSP-C surfactant (25 and 100 mg PL per kg body weight) combined with the PDE-4 inhibitor at a dose of 6.0 mg/kg body weight. The different groups were compared with respect to improving oxygenation and histopathologic changes, e.g., hyaline membrane (HM) formation. Both doses of rSP-C surfactant improved oxygenation while even this high dose of the PDE-4 inhibitor alone did not influence oxygenation compared with untreated control animals. Addition of the PDE-4 inhibitor led to improved oxygenation based on both doses of rSP-C surfactant. The PDE-4 inhibitor alone prevented further HM formation and infiltration of neutrophil leukocytes. The rSP-C surfactant was able to prevent further HM formation. Based on both doses of rSP-C surfactant, addition of the PDE-4 inhibitor showed additional effects on oxygenation and inhibition of HM formation. The effects of combined treatment were superior to each treatment alone, leading to the conclusion that a rSP-C surfactant containing a PDE-4 inhibitor may act synergistically in this animal model of acute lung injury. We conclude that combined treatment with rSP-C surfactant and a PDE-4 inhibitor may be an effective treatment for patients with acute lung injury.

DNA vaccination with both the haemagglutinin and fusion proteins but not the nucleocapsid protein protects against experimental measles virus infection.

Plasmids that expressed the nucleocapsid, haemagglutinin and fusion proteins of measles virus (MV) were used to immunize cotton rats (Sigmodon hispidus) against intranasal MV infection. After immunization with all three plasmids, T cell responses and MV-specific antibodies were induced. A reduction in virus titre was observed in lung tissue from animals immunized with plasmids expressing the viral glycoproteins. Histologically, however, a moderate peribronchitis was observed after immunization with the plasmid expressing the fusion protein whereas, after immunization with plasmids expressing haemagglutinin or both glycoproteins, only mild or focal peribronchitis was seen. Immunization with the nucleocapsid did not reduce virus titres, probably because of the failure to induce neutralizing antibodies. A disadvantage of plasmid immunization was its inefficacy in the presence of MV-specific 'maternal' antibodies. This indicates that genetic immunization has to be improved to be a useful alternative vaccine against measles.

A rat model of acute respiratory distress syndrome (ARDS) Part 2, influence of lavage volume, lavage repetition, and therapeutic treatment with rSP-C surfactant.

UNLABELLED: The influence of lavage volume, and lavage repetition with physiological saline solution (groups 1-3: 3x4, 4x4, 5x4, groups 7-9: 3x8, 5x8, 7x8, mL per animal) was studied in a rat lung lavage model of the acute respiratory distress syndrome (ARDS). Anesthetized and tracheotomized rats (12 rats/group) were pressure-controlled ventilated with 100% oxygen at a respiratory rate of 30 breaths/min, inspiration: expiration ratio of 1:2, peak inspiratory pressure of 28 cm H2O at positive end-expiratory pressure of 8 cm H2O during the whole experimental period. To investigate the influence of therapeutic treatment, a recombinant surfactant protein C (rSP-C) containing surfactant was used. Therefore, rats which received a lavage of 4x4 mL per animal (groups 4 to 6) or 7x8 mL per animal (groups 10-12) were treated intratracheally with surfactant doses of 12.5, 25, or 100 mg phospholipids (PL) per kg body weight (bw). In all groups, partial arterial oxygen pressures (PaO2, mm Hg) and partial arterial carbon dioxide pressures (PaCO2, mm Hg) were determined 30 min before, directly after, and 5, 30, 60, 90, 120, 150, 180, and 210 min after the last lavage. Additionally, animals were euthanized 210 min after the last lavage for semiquantitative histopathological grading of coded lung slides. Grading was performed with respect to the severity of hyaline membrane formation (HM), margination and infiltration of polymorphonuclear neutrophil leukocytes (PMNL) into the lung alveoli and interstitial and intraalveolar edema (E). The intrapulmonary distribution of intratracheally applied rSP-C was estimated in selected lung slides stained with polyclonal anti-rSP-C antibody and was compared to unlavaged control rats and unlavaged rats which received 100 mg/kg bw rSP-C. The repetitive lavage depleted the lung from its natural surfactant resources leading to a pathophysiological cascade similar to that of the acute respiratory distress syndrome. PaO2 levels and HM formation showed a lavage-induced decrease. Both changes were significantly dependent on the repetition and volume of the lavage; however, the parameters PMNL and E did not show such a dependence. Treatment with rSP-C surfactant significantly improved oxygenation and reduced HM-formation in a dose-dependent manner independent from the lavage volume. All doses of rSP-C surfactant showed no clear influence on the parameters PMNL and E independently from the lavage volume. In lavaged rat lungs (ARDS-model), the exogenously applied rSP-C was distributed homogeneously along the alveolar lining. Unlavaged rats that received a similar dose of rSP-C showed a marked inhomogeneous extracellular distribution, mainly associated with larger bronchi, while the type II pneumocytes were stained positively in unlavaged control and unlavaged rSP-C treated rats. CONCLUSION: This model mimics very closely the wide spectrum of the clinical situation of human acute lung injury (ALI) because the variation of lavage volume and repetition lead to reproducible different severity grades and states of ALI. The significant reduction of pathognomic changes due to treatment with rSP-C surfactant showed that this is a useful model to estimate the influence of therapeutic concepts in ALI and ARDS.

Concanavalin A hepatotoxicity in mice: tumor necrosis factor-mediated organ failure independent of caspase-3-like protease activation.

Several models of tumor necrosis factor (TNF)/TNF-receptor 1 (TNF-R1)-dependent liver injury in mice were investigated with respect to caspase-3-like protease activation representing a pivotal mechanism of apoptotic cell death. Injection of TNF or T-cell-activating agents (i.e., agonistic anti-CD3 antibody or staphylococcal enterotoxin B [SEB]) into galactosamine (GalN)-sensitized mice caused TNF/TNF-R1-dependent liver injury. Intravenous concanavalin A (Con A) alone induced TNF-mediated hepatotoxicity dependent on both TNF-R1 and TNF-R2. Hepatic caspase-3-like proteases were activated in GalN/TNF, GalN/anti-CD3, or GalN/SEB-treated mice, but not in Con A-treated mice. Consistently, the broad-spectrum caspase inhibitor, benzoyloxycarbonyl-val-ala-asp-fluoromethylketone (zVADfmk), prevented TNF-mediated hepatotoxicity in all GalN-dependent models, but failed to protect against Con A. Under transcriptional arrest, however, Con A induced TNF-R1-dependent, but not TNF-R2-dependent, activation of caspase-3-like proteases, and zVADfmk prevented animals from Con A-mediated liver injury under this condition. Histological analysis revealed distinct differences between Con A- and GalN/Con A-induced liver injury regarding apoptotic morphology of hepatocytes. We conclude that impaired transcription induces a switch of Con A hepatotoxicity toward a caspase-3-like protease-dependent pathway. The observation that the functional state of the transcriptional machinery decides whether TNF-driven hepatocyte apoptosis involves activation of caspase-3-like proteases or alternative signaling pathways in vivo might be of relevance for the immunopathology of the liver.

Effects of early treatment with rSP-C surfactant on oxygenation and histology in rats with acute lung injury.

Surfactant treatment in patients with acute respiratory distress syndrome (ARDS) may be a promising treatment strategy. The aim of this study was to investigate whether addition of a recombinant surfactant protein C (rSP-C) to a plain phospholipid (PL) surfactant (PL surfactant) can result in activity comparable to commercially available surfactant preparations (Alveofact and bLES) which contain surfactant protein B and C. In this investigation dose-response comparisons of four surfactants were performed in an animal model of ARDS induced by total lung lavage. The surfactants were given shortly (;10 min) after the last lavage. The effects of surfactant treatment were compared with respect to improve oxygenation and to prevent histopathological changes, such as hyaline membrane formation. The surfactants were compared to lavaged, untreated controls. The surfactants were administered at doses of 25, 50 and 100 mg total amount of phospholipids/kg body weight. At 120 min after early treatment, all three doses of rSP-C surfactant showed statistically significant higher improvements in oxygenation than PL surfactant. This improvement was comparable to bLES and superior to Alveofact. The rSP-C surfactant showed the most prominent effect on preventing hyaline membrane formation. It was again superior to PL surfactant and comparable to bLES. It is concluded that addition of rSP-C enhances the activity of a pure PL surfactant. The rSP-C surfactant showed comparable or even superior activity to bovine-derived surfactant preparations containing both, SP-B and SP-C.

Ito cell tumor: immunohistochemical investigations of a rare lesion in the liver of mice.

In 2 lifespan transgeneration experiments using a total of 4,682 CBA/J mice, we observed uncommon lipomatous lesions in the livers of 8 mice independent of the treatment. Macroscopically, the lesions were described as pale white areas (2) or nodules (6) during necropsy. The lesions ranged from 1 to 15 mm in diameter. Microscopically, the lesions consisted of nodular aggregations of round to spindle-shaped cells that partly caused distinct compression of the adjacent hepatic parenchyma. The tumor cells were smaller than hepatocytes and had dark oval nuclei. Many of the more spherical cells contained clear vacuoles of various sizes, which were shown to be lipid droplets by oil red O staining. In addition to Gomori's silver and Masson's trichrome staining, several immunohistochemical stains were used to characterize the origin of the proliferating cells. Tumor cells were labeled by vimentin, actin, desmin, and proliferating cell nuclear antigen. The 2 cell phenotypes showed similar staining characteristics. Increased amounts of laminin and tenascin, 2 extracellular matrix proteins of the liver, were detected within these neoplasms. Summarizing, we suggest that these tumors are of Ito cell origin.

RITA/Registry of Industrial Toxicology Animal data: a comparative immunohistochemical study of 77 islet cell carcinomas in Sprague-Dawley and Wistar rats using antibodies against insulin, glucagon, somatostatin and gastrin.

UNLABELLED: The objective of this study was to investigate spontaneous islet cell carcinomas with particular reference to possible existing strain differences between Sprague Dawley (SD) and Wistar (W) rats in incidence and immunohistochemical staining pattern. Secondly the occurrence of somatostatin and/or gastrin-positive islet cell tumors should be tested. Islet cell adenocarcinomas (34 from SD, 43 from W-rats) were selected from the RITA-data base and company in-house data base out of an animal pool of 3915 (1681 SD, 2234 W-rats). They were untreated or sham-treated (vehicle) control animals from carcinogenicity studies and whole life-span experiments. Islet cell carcinomas occurred in a higher incidence in male rats (2.98% for SD, 3.23% for W) than in female rats (1.07% for SD, 0.63% for W). All specimens were immunohistologically stained with antibodies against insulin, glucagon, somatostatin and gastrin and, selected specimens with additional antibodies (pancreatic polypeptide, lipase, chymotrypsin, S100-protein, actin and cytokeratin). 94% (SD) and 93% (W), respectively, were insulin-positive and the mean staining intensity (on a scale ranging from 0-4) for insulin was 3.58 (SD) versus 3.37 (W). This high insulin staining incidence and intensity characterized most islet cell carcinomas as malignant insulinomas. 24% (SD) and 37% (W), respectively, were glucagon-positive. Except two tumors in W-rats with a focal strong glucagon expression, the mean staining intensity for glucagon was low (0.38 SD, 0.72 W). 38% (SD) and 44% (W), respectively, were somatostatin-positive, but except for five cases having a focal to multifocal, moderate to marked staining, only a few tumor cells were positive for somatostatin in the other cases and the mean staining intensity for somatostatin was low (0.50 SD, 0.84 W). 6% (SD) and 23% (W), respectively, were gastrin-positive, but only one case of a male Wistar rat exhibited a focal strong staining in parts of the tumor. The other cases showed only a few tumor cells which were positive for gastrin. The mean staining intensity for gastrin was low (0.06 SD, 0.35 W). In all tumors with marked glucagon, somatostatin or gastrin expression, the immunostaining for insulin was still predominating. Thus, insulin was the major hormone produced by most of the tumor cells. Five out of 77 tumors evaluated were immunohistologically negative with all applied antibodies. CONCLUSION: This study presents the first immunohistochemical survey on spontaneous islet cell carcinomas in SD and Wistar rats stained with antibodies against the endocrine pancreas hormones insulin, glucagon, somatostatin and gastrin. No major differences in incidence or immunohistochemical staining pattern between SD and W-rats could be detected. In contrast to SD rats, Wistar rats had multihormonal coexpression in 16.3%. The multihormonal appearance of the neoplasms is well comparable with the findings in other animal species and human insulinomas. Moreover, this is the first study in rats which reports five cases with a marked co-expression of somatostatin and one case with marked focal co-expression of gastrin in malignant islet cell adenocarcinomas.

Intratracheally applied rSP-C surfactant exhibits no anaphylactic shock reactions in a guinea pig model of acute lung hypersensitivity.

The effect of the intratracheal administration of the recombinant SP-C surfactant apoprotein (rSP-C) with phospholipids (PL) in comparison to an ovalbumin induced anaphylactic shock reaction was studied in guinea pigs lungs. Narcotized guinea pigs were challenged by intratracheal administration on test day 24/25 once with a suspension of rSP-C/PL (reconstituted suspension). These animals were priorily sensitized on test day 1, 3 and 5 intraperitoneally with rSP-C/PL suspension or with Ovalbumin (OV) respectively. The following groups were used to assess the anaphylactic lung shock symptoms: group 1: positive control, 1 mg/kg OV protein, 2 ml/kg application volume, (Appl. vol.), N: 5 animals; group 2: 1 mg rSP-C/50 mg PL/0.5 ml/kg Appl. vol., N: 10; group 3: 2 mg rSP-C/100 mg PL/1.0 ml/kg Appl. vol., N: 10; group 4: 4 mg rSP-C/200 mg PL/2.0 ml/kg Appl. vol., N: 10. Clinical signs, mortality, lung weights and histopathological changes were evaluated. Additionally the lungs were investigated immunohistologically with polyclonal antibodies against rSP-C to determine the pulmonary distribution of the intratracheal applied rSP-C. In the OV-treated positive control group, all animals died within 4 minutes after intratracheal challenge, while only 1 animal of group 4 died probably due to an narcosis related respiratory arrest. In the rSP-C/PL treated groups, the lung weights showed a dose-related increase, but nevertheless all these rSP-C-treated groups showed a significant lower lung weight in comparison to the OV treated positive control group. The histopathology assessment of the lungs in the OV-treated animals revealed a severe generalised bronchoconstriction and a hyperemia in connection with a slight interstitial edema in all five animals. The rSP-C/PL-treated animals, which were sacrificed after 3 days, showed no bronchoconstriction but a slight increase in the severity of bronchus-associated infiltration with eosinophilic granulocytes and in the formation of peripheral emphysema, but with no dose-dependency. A slight dose-dependent increase in the deposition of peribronchiolar eosinophilic foreign material was evident. In contrast to this, the number of lipid-laden alveolar macrophages seemed to decrease with increasing doses of rSP-C/PL. The immunohistological investigation with a polyclonal antibody against rSP-C showed an intraalveolar distribution of the intratracheally applied rSP-C which is mainly located in the peribronchiolar alveolar parenchyma. A rSP-C-positive staining was visible within the cytoplasm of alveolar histiocytes, type II pneumocytes and also as an extracellularly rim along the alveolar walls. The polyclonal antibody showed no cross reaction with natural occuring SP-C-protein of the guinea pigs. We conclude that the intratracheal application of the rSP-C surfactant containing phospholipids (PL) exhibits no significant risk of an anaphylactic shock reaction in this guinea pig lung hypersensitivity model. The immunohistological investigation with polyclonal antibodies against rSP-C demonstrated clearly the distribution of intratracheal applied material in this toxicological animal model.

Dexamethasone enhances the activity of rSP-C surfactant but not of exosurf in a rat model of the acute lung injury.

The possible enhancement of surfactant activity by pretreatment with a glucocorticosteroid (dexamethasone) was investigated in a rat lung lavage model of acute lung injury. Animals received a dose of dexamethasone (10 mg/kg i.p.) prior to the protein-free surfactant preparation Exosurf (pure phospholipids containing surfactant, Wellcome GmbH, Burgwedel, Germany) and a rSP-C based surfactant, respectively. Both surfactants were intratracheally instilled at doses of 25 and 100 mg phospholipids per kg body weight and were compared with pretreatment with dexamethasone at each dose level. These groups were also compared with untreated controls and to pretreatment with dexamethasone alone with respect to improvements in oxygenation, to inhibition of infiltration of polymorphonuclear neutrophil leukocytes (PMNL) and to influence formation of hyaline membranes. Dexamethasone alone had no influence on the reduced PaO(2) but reduced the infiltration of PMNL and the formation of hyaline membranes. Dexamethasone improved the oxygenation at both doses of rSP-C surfactant. At the low dose of rSP-C surfactant there were additional effects detectable with regard to histopathologic improvements. In contrast, dexamethasone had no additional effect on oxygenation and formation of hyaline membranes when combined with Exosurf. Only the infiltration of PMNL was decreased by combined treatment with dexamethasone and Exosurf. The effect was comparable to that of pretreatment with dexamethasone alone. In this animal model, pretreatment with dexamethasone showed additional effects on rSP-C surfactant that were superior to each treatment alone. From the comparison of rSP-C surfactant with the synthetic surfactant preparation Exosurf, we conclude that the activity of Exosurf cannot be improved substantially by additional pretreatment with drugs like glucocorticosteroids.

Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome.

1. In a previous paper we showed that an SP-C containing surfactant preparation has similar activity as bovine-derived surfactants in a rat lung lavage model of the adult respiratory distress syndrome. In this study surfactant was given ten minutes after the last lavage (early treatment). In the present investigation we were interested how different surfactant preparations behave when they are administered 1 h after the last lavage (late treatment). 2. Four protein containing surfactants (rSP-C surfactant, bLES, Infasurf and Survanta) were compared with three protein-free surfactants (ALEC, Exosurf and the phospholipid (PL) mixture of the rSP-C surfactant termed PL surfactant) with respect to their ability to improve gas exchange in this more stringent model when surfactant is given one hour after the last lavage. For better comparison of the surfactants the doses were related to phospholipids. The surfactants were given at doses of 25, 50 and 100 mg kg(-1) body weight. The surfactants were compared to an untreated control group that was only ventilated for the whole experimental period. 3. Tracheotomized rats (8-12 per dose and surfactant) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min(-1), inspiration expiration ratio of 1:2, peak inspiratory pressure of 28 cmH2O at positive endexpiratory pressure (PEEP) of 8 cmH2O. Animals were ventilated for one hour after the last lavage and thereafter the surfactants were intratracheally instilled. During the whole experimental period the ventilation was not changed. 4. Partial arterial oxygen pressures (PaO2, mmHg) at 30 min and 120 min after treatment were used for statistical comparison. All protein containing surfactants caused a dose-dependent increase of the reduced PaO2 values at 30 min after treatment. The protein-free surfactants showed only weak dose-dependent increase in PaO2 values at this time. This difference between the protein-containing and the protein-free surfactants was even more pronounced when comparing the PaO2 values at 120 min after treatment. Only rSP-C surfactant, bLES and Infasurf showed a dose-dependent increase in PaO2 at this time. 5. With this animal model of late treatment it is possible even to differentiate between bovine derived surfactants. The differences between protein-containing and protein-free surfactants become even more pronounced. From the comparison of rSP-C surfactant with bovine-derived surfactants and the PL surfactant without rSP-C, it can be concluded that addition of rSP-C is sufficient to achieve the same activity as that of natural surfactants.

Effects of rSP-C surfactant on oxygenation and histology in a rat-lung-lavage model of acute lung injury.

We have tested two surfactant preparations with the same phospholipid (PL) composition, containing recombinant surfactant protein-C (rSP-C surfactant) and without SP-C (plain PL surfactant). The effects of rSP-C surfactant were compared with the bovine-derived surfactant preparations Alveofact, bLES, and Infasurf in a lung lavage model, with surfactant given 1 h after the last lavage. The effects of surfactant treatment on histopathologic changes (e.g., hyaline-membrane formation) and improvement of oxygenation were compared with changes in untreated controls. The surfactants were given in doses of 25, 50, and 100 mg PL/kg body weight. At 120 min after treatment, only the protein-containing surfactants showed a statistically significant increasing dose dependence with respect to improving oxygenation. The values were 318 +/- 120 mm Hg, 443 +/- 58 mm Hg, and 480 +/- 43 mm Hg (mean +/- SD) for the three doses of rSP-C surfactant and 105 +/- 81 mm Hg, 100 +/- 69 mm Hg, and 131 +/- 108 mm Hg for the three doses of PL surfactant. The respective values for Alveofact were 104 +/- 81 mm Hg, 105 +/- 93 mm Hg, and 260 +/- 143 mm Hg; for bLES 373 +/- 138 mm Hg, 441 +/- 88 mm Hg, and 467 +/- 43 mm Hg; and for Infasurf 146 +/- 96 mm Hg, 284 +/- 178 mm Hg, and 436 +/- 70 mm Hg. The oxygen values of controls remained low, at 74 +/- 46 mm Hg. Only the protein-containing surfactants dose-dependently inhibited the formation of hyaline membranes. We conclude that rSP-C surfactant is at least as effective as bovine-derived surfactants. Furthermore, the data imply that the difference between plain PL surfactant preparations and bovine-derived surfactant preparations containing both SP-B and SP-C can be overcome by addition of SP-C.

Pathology of the oropharyngeal cavity in six strains of rats: predisposition of Fischer 344 rats for inflammatory and degenerative changes.

The sagittolongitudinal histopathological evaluation of the oropharyngeal cavity in 6 strains of rats revealed a clear predisposition of the Fischer 344 strain for degenerative and inflammatory lesions in this region. A degeneration and calcification predominantly of the laryngeal cartilage is evident in 72%, granulomas of the seromucinous glands in 38%, and an inflammation with bacterial colonies or plant material in the basal part of the laryngotracheal junction in 26% of the Fischer 344 rats. The incidences of these types of lesions were negligible in the other rat strains evaluated. The comparison of control Fischer 344 rats from 2 carcinogenicity studies (untreated group; gavaged group with water, pH 10; gavaged group with propylene glycol/methocel) showed an influence of the gavage technique with the solvent on the survival rate in the female Fischer 344 rats. The lower survival rate was accompanied by the occurrence of food impaction in the oropharyngeal cavity. This increased mortality in female Fischer 344 rats was also associated with a degeneration of the laryngeal cartilages, whereas the formation of granulomas and inflammation seemed to have no direct relationship to the mortality. The gavage technique with the applied solvent was an enhancing factor in the female Fischer 344 rats on mortality, on cartilage degeneration, but not on inflammatory changes. In contrast to the females, male Fischer 344 rats had a higher incidence of granulomas and inflammation, which, however, did not influence the survival rate. In conclusion, Fischer 344 rats, especially the female sex, exposed to chronic daily irritation by gavage, are predisposed to have a high mortality rate in chronic toxicologic tests. An explanation lies in the disposition of the Fischer 344 rat to a high incidence of cartilage degeneration, which presumably leads to a dysfunction of the larynx.

A rat model of acute respiratory distress syndrome (ARDS): Part 1. Time dependency of histological and pathological changes.

The time course of histopathological changes in a rat lung lavage model of the acute respiratory distress syndrome (ARDS) was analyzed by sacrificing animals 10, 30, 60, 180, and 210 min after the last lung parenchyma lavage which was performed with physiological saline solution. This lavage depleted the lung from its natural surfactant resources leading into a pathophysiological cascade similar to that of the acute respiratory distress syndrome. Tracheotomized rats (12 animals per time point) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths/min, inspiration-expiration ratio of 1:2, peak inspiratory pressure of 28 cm H2O at positive end-expiratory pressure (PEEP) of 8 cm H2O. During the whole experimental period, the ventilation was not changed. Blood gases (partial arterial oxygen pressures [PaO2, mmHg] and partial arterial carbon dioxide pressures [PaCO2, mmHg]) were estimated before, directly after, and 10, 30, 60, 90, 120, 150, 180, and 210 min after the last lavage. For grading lung lavage-induced histopathological changes associated with the time-dependent development of ARDS, slides were coded and evaluated without any knowledge of the sacrifice time. A semiquantitative grading was performed with respect to the severity of the following parameters: hyaline membrane formation (HM), interstitial and intraalveolar edema edema (E), and margination and infiltration of polymorphonuclear neutrophil leukocytes (PMNL) into the lung alveoli. The severity of these parameters showed a time-dependent increase after the last lavage. This was accompanied by a time-dependent decrease in partial arterial oxygen pressure (PaO2) values during the early postlavage period (up to 30 min). Thereafter, PaO2 levels remained fairly stable. The severity of intraalveolar and/or perivascular hemorrhages within the lung was not time dependent. The rat lavage model shows similarities to the pathophysiological sequelae occuring during the acute phase of the acute respiratory distress syndrome in humans. Most of the characteristic pathognomic histological changes seen in humans can be observed in this lung lavage model. This ARDS model is brief and easy in its experimental design, showed a good and homogeneous reproducibility of pathophysiological and histopathological parameters, and is therefore a useful model to estimate the influence of therapeutic pharmacological treatments of ARDS.

Tumor necrosis factor-induced hepatic DNA fragmentation as an early marker of T cell-dependent liver injury in mice.

BACKGROUND & AIMS: The injection of mice with anti-CD3 monoclonal antibody causes activation of T lymphocytes and leads to a lethal shock syndrome. The aim of this study was to investigate T cell-dependent, cytokine-mediated target cell death that leads to organ injury. METHODS: Anti-CD3 monoclonal antibody or staphylococcal enterotoxin B was injected into mice sensitized by D-galactosamine. Liver injury was assessed biochemically and histologically, and circulating cytokines were determined. RESULTS: Mice sensitized with D-galactosamine developed severe liver injury within 8 hours after injection of anti-CD3 monoclonal antibody or staphylococcal enterotoxin B. Apoptotic bodies and chromatin condensation were detectable 5 hours after anti-CD3 monoclonal antibody challenge. DNA fragmentation in the liver preceded the increase in plasma alanine aminotransferase activity. Anti-CD3 monoclonal antibody induced the release of tumor necrosis factor and other cytokines. Passive immunization against tumor necrosis factor or pretreatment with immunosuppressive drugs protected mice from liver injury. Liver injury associated with apoptotic cell death and DNA fragmentation was also noted in D-galactosamine-sensitized mice injected with staphylococcal enterotoxin B. CONCLUSIONS: Tumor necrosis factor-induced hepatic apoptosis followed by necrosis may represent a general pathomechanism of T-cell shock models using D-galactosamine-sensitized mice.