it_particles_inflammation

Size effect of intratracheally instilled particles on pulmonary inflammation and vascular thrombosis.

Nemmar, A., Hoylaerts, M.F., Hoet, P.H.M., Vermylen, J., Nemery, B. Tox. Appl. Pharm. 186: 38-45, 2000.

This work begins to explain recent findings of human epidemiological studies showing that exposure to PM₁₀ is related to both respiratory disorders and cardiovascular problems. One theory explaining this finding is that inflammatory mediators released in the lungs after breathing PM₁₀ and UFP are involved in the etiology of the cardiovascular effects. UFP have been shown to be even more inflammatory than PM₁₀ when measured per unit mass.

The purpose of this study was to examine the acute effects of different sizes of small charged particles administered via intratracheal instillation into hamsters’ lungs. The effects studied were 1) pulmonary inflammation as measured by BAL and 2) thrombogenesis in the femoral vein as well as the lung. Thrombogenesis in the lung was measured by a stain for platelets along with histology. A link between particle induced inflammation and thrombosis was looked for as well. In earlier studies, this group of investigators showed that ultrafine particles crossed the alveolar-capillary barrier in hamsters in less than 1 hr. These small particles given both i.v. and i.t. affected thrombus formation in the hamster via platelet activation. The small particles used were amine modified polystyrene particles.

Materials and Methods:
Particles and treatment groups: Both 60 nm and 400 nm polystyrene particles were used. The 60 nm particles were either unmodified or substituted with amine or carboxylate groups. The 400 nm particles were modified with amine groups only. All particles were given in saline solution by an intratracheal instillation technique involving shaving the trachea; the specific technique is not elaborated upon. The experimental animals were both male and female hamsters. Specific numbers of each sex were not specified.

The treatment groups were:
1)     Controls
2)     60 nm Carboxylate UFP’s
        a.     Unmodified 500 ug/animal
        b.     Carboxylate 500 ug/animal
        c.      Amine 5 ug/animal
        d.     Amine 50 ug/animal
        e.     Amine 500 ug/animal
3)     400 nm Carboxylate Particles
        a.     Amine 500 ug/animal

Peripheral thrombosis was measured by giving Rose Bengal via a venous catheter into the right jugular vein 10” after the particle instillation. The right femoral vein was exposed and mounted on a transilluminator. After the Rose Bengal injection, green light was used to irradiate the femoral vein. This technique results in a mild thrombosis the formation of which was measured for 40” with a microscope and recorded with a camera. Although not specified, the animals must have still been anesthetized from the initial i.t. instillation anesthesia during this procedure. After the recording of the femoral vein all animals were killed at one hour post-instillation.

Histology, Measures of Inflammation: Seven um frozen sections were prepared from lung tissue. They were either stained either with Hematoxylin and Eosin or murine anti-platelet GPIbµ antibody G28E5 which cross-reacts with hamster GPIbµ, thus marking platelets. Protein, LDH, and histamine were measured from BAL obtained by standard procedures. Platelet function was measured in venous blood with a Platelet Function Analyser PFA-100. The data are all presented as the means +/- SEM and groups were compared with one-way ANOVA followed by Dunnett’s multiple range test.

Results: Effect of intratracheal instillation of particles on thrombus formation: Thrombi formation are measured and expressed as the light intensity in A.U.at the femoral artery after photochemical injury. The authors state that, of all treatment groups defined above, only amine-UFP’s significantly enhanced thrombus generation. This increase was found at doses of 500 and 50 ug of the 60 nm particles/animal. Although there appears to be an increase with 500 of the 400 nm particles, it is not significant.

Histology: Evidence that particle instillation causes pulmonary thrombosis was seen in lungs of animals treated with 500 ug amine-UFP’s. This evidence was the appearance of pulmonary thrombi positively stained for G28E5, the platelet marker.

Effect or particles on BAL: Amine-UFP’s caused a significant PMN influx with 500 and 5 ug of the 60 nm particles/animal. There was an influx of neutrophils in the group receiving 500 ug of the 400 nm particles. Protein was increased in the group receiving 500 ug amine UFP’s as well as the group receiving 500 ug of the 400 nm amine particles. Significant increases in total BAL protein were observed in all three of the amine particles that were 60 nm but not in the 400 nm particle group.

Discussion: The authors spend much of the discussion explaining the rationale for the study design. They explain their investigation for links between local and peripheral thrombotic events as an attempt to verify the currently common assumption that cardiovascular effects after inhalation of PM10 are due to inflammatory mediators released by the lung. They found that positively charged UFP enhanced vascular thrombosis within one hour after particle instillation into the lung. The same size particles with neutral or negative charges and larger particles did not have this effect.

The authors believe their model is an excellent tool for examining potential anti-thrombotic and pro-thrombotic agents. They compare these results with those found when they administered particles intravenously. They emphasize that these data confirm findings in rodents showing UFP’’s administered to the lung cause more inflammation than do larger particles of the same total mass. The authors also feel it is significant that UF charged particles caused thrombophilia whereas negative and neutral particles do not. The implication of this finding is that both particle size and surface charge somehow determine the development of the thrombotic state.

Finally, and perhaps most important, the authors present their evidence that pulmonary inflammation and peripheral thombotic events may not be totally coupled. They cite the evidence provided by this experiment that links thrombosis and inflammatory changes in BAL. That is, the negative and neutral UFP that did not cause increased thrombosis did not cause pulmonary inflammation. But UF positive particles cause both injury and inflammation in the lungs. However the highest amount in inflammation (from 500 ug positive UFP/animal did not correlate with the greatest amount of thrombosis. The authors did not think that the enhanced thrombotic complications in the 50 ug/animal group can be due to pulmonary inflammation alone. Rather they suggest that peripheral thrombotic events may be caused by direct migration of UFPs and activation of circulating platelets.

Conclusion: The authors conclude that particle-induced lung inflammation and thrombogenesis are not necessarily linked together. The two processes may be partly uncoupled. Pulmonary inflammation if maximal with an hour after i.t. administration of positive UFP and 400 nm particles. Positive UFP’s but not 400-nm particles enhance thrombosis.

By: Susan G. Shami, ScD, Senior Science Editor
http://www.Susanshami.com/index.html

Editorial note: This is a very cleverly designed experiment for the purpose of answered the questions asked. However, there is a question regarding the validity of the model used for administration of particles to the lung. Intratracheal instillation is very different from inhalation. It is used infrequently now compared to 20 years ago. Most studies using the i.t. technique do not have time points as close as an hour after instillation. At this point in time, the lungs are still affected by the saline of the instillation, anesthesia, and the particles administered. Several studies have shown that saline instillation alone causes inflammation, increased cell proliferation, and possibly epithelial cell injury (1). In addition one must consider the fact that the animals are apparently anesthetized for the entire time of the experiment. Although the authors do not state this, there seems no other way to examine the femoral vein unless the animal is restrained. Hence respiration is not as it would be in a “normal animal”. Finally simply having the animals in the supine position required of the i.t. technique is not normal either. The model, in fact, seems to simulate a human immobilized briefly and possibly with mild pulmonary edema. Obviously the techniques used do not lend themselves to a more realistic situation such as would occur with inhalation of particles. It seems that the authors should have addressed this i.t. instillation model in their discussion.
The other point which must be emphasized is that the animals used were both male and female hamsters. After this statement, the reader does not know how the sexes were divided or put into treatment groups. Now that gender differences are becoming very obvious and important in studies, it is difficult to imagine why a single sex was not used. Omitting females could have reduced variablility significantly. This was the reason animal studies have always favored males. Another paper reviewed in this issue of the newsletter shows that female mice are more sensitive to acute toxicity of a chemical than are males (Gender differences in naphthalene metabolism and acute toxicity in lungs). In addition several recent reviews (Gender and enhanced sensitivity to odors, Odor, Irritation and Perception of Health Risk ) show gender differences with regard to olfactory stimuli. By: Susan G. Shami, ScD 21 May 03

1) Shami, Thibodeau, Kennedy, Little, Cancer Research 42, 1405-1411, April 1982. Proliferative and Morphological Changes in the Pulmonary Epithelium of the Syrian Golden Hamster during Carcinogenesis initiated by ²¹⁰Po alpha-radiation.