"Trinoculaire" de Microscopies Electroniques, Lausanne, juin 1995 231

I N VIVO A N D I N V IT RO E F F E C T OF K I L L E D

P R O P I O N I B A C T E R I L I M A V I D U M ON R A T K U P P F E R CELLS.

GENDRALILT J.Louis. }tEUSS Rachel, DHOU[B Mounir *, LUGNIER Alain *

Z,-lho:;7/<)l?'e (t¢ V/:oh)~ve Ef I,~fUA)/II U74, Facultd de/l.[dc/eci::e, 5?r, Tx'bour~o,

"Zai, or, v:o:;,-e (le 7owk'olo<.,:7= Fo::d, Tx#ei::,71e e: d'Ec'oro.ricolc~ie UL~Stra_~'bouG,':.

Killed P:opio::i&~cteriu::~ ,Tv:?/u:/:(PPA) has been administered to both Wistar

rats and isolated rat KNpffer cells (KC), the so-called macrophages of the liver.

PPA bacteria were cultivated in brain-hear t infusion under controlled

anaerobiosis for 72 hours at 37°C. Subsequently, the opaque suspension

obtained in 2ml of 0.9% NaCI was submitted to potential bactericidal conditions

(beat treatment). Male Wistar rats (250g) received 30mg/Kg body weight of

killed PPA for 4 to 192 hours by intraperitoneaf or intravenous rome. The livers

were fixed by perfusion wilb 2.5% glutaraldehyde in 0.1M Na cacodylate

buffer. For i:2 vi:ro studies, the KC were isolated by collagenase dissociation of

the liver and centrifugal elutriation according to the method previously

described (Kirn A. et al. (19821 Hepato/ogy, 2, 216-222) and cultured for

24 hours. They were then treated for one to fonr days with different dilutions of

a suspension of 1.75rng/ml killed PPA. The cells were fixed ill 2.5%

glalaraldehyde in 0.1M Na cacodylate buffer. In order to characterize the KC,

IBm-sized latex beads were injected via the caudal vein and the presence of

endogenous peroxidase was checked. For TEM the samples were post-fixed in

OsO4 and embedded in I.X I I2 . For SEM tile samples were dried with

bexamethyldisilazaue and coated with gold-palladium.

After intraperitoneal injection of killed PPA, the livers displayed a rather

normal appearance with bacteria being observed in typical KC. Some large cells

occnpyiug alurost all tile hn/lall of the sinnsoid were seen from place to place.

They displayed processes often interulingled with blood cells and numerous

vacnoles. Those cells could be nllambiguotlsly interpreted as KC. Their number

increased with tinle and they were also present after intravenous injection of

PPA. Interestingly, some mitotic figures could be observed in hepatocytes and in

KC as well.

After adminislration of killed PPA to KC cultures, numerous bacteria were

found inside phagocytic vacuoles where degradation occurred.

KC were idenlified in the rat liver to be responsible for the uptake of PPA.

These findings are consistent with the hypothesis that killed PPA may promote

hepatocellnlar damage.

STRUCTURE AND CYTOLOGY OF THE OLFACTIVE BULB OF NORMAL AND "STAGGERER" MUTANT MICE;

BRIDE Michelle, BRIDE Jaequeline, MATH Francois. Laboratoire de Neurosciences, Facultd des Sciences, BESANCON, France;

The "Staggerer" mouse mutation is known for a long time for its cerebellar effects. Besides Iocomotory impairment, other comportmental troubles in reproductive behaviour have been described, due to a defect in male-female recognition mechanism. These may depend on olfactive lesions. Could the defects in the structural organization also affect the olfaetive bulb?

To answer this question, structural and cytological observations were performed on normal and mutant adult C57/B6 olfactive bulbs. In both cases, the different zones usually distinguished (i.e. nervous, glomerular, plexiform, mitral, granular) are recognizable, while being less thick in the mutant.

In spite of the fact that the different cell types are not easily discriminated on the pure basis of their structural characteristics, our observations speak for a preservation of the cell specificity in the mutant. The bulb organization is however undoubtedly impaired in the mutant. Alterations are revealed by a far less tight architectony, due to a reduced number of neurones. Deutoneurones (mitral cells) and interneurones (as the periglomerular cells) are particularly less abundant (-20%) than in controls, while the glomerular zone size is less developed (-10%). As a result, cell processes emanating from the glial cells tend to invade in an obvious manner the intercellular spaces.

Electrophysiological analyses perfectly agree with the olfactive defects mentioned for the mutant mice. Electrocorticographical recordings practized on olfactive bulbs display analogies for both control and mutant mice, but, in the latter, burst frequency is weaker and their duration longer than in controls.

Moreover, the evoked potentials, which represent an answer to a specific odor, induce in mutants graph modifications which agree perfectly with the mentioned structural observations; these modifications demonstrate a decrease of both the ability to concentrate informations in the glomerular zone and the capacity to integrate them in the mitral cells. Due to the particular phenomena which affect neurogenesis in the olfactive system, the two experimental models and the ways we choose for their study appear to be promising for further research on the mechanisms implied in the synaptical reorganization which takes place in the olfactive bulb.

C H A R A C T E R I Z A T I O N OF LASER INDUCED LESIONS IN RABBIT RETINA BY COMBINED SCANNING CONFOCAL AND TRANSMISSION ELECTRON MICROSCOPY. LE NAOUR Hdl~.ne, FRITSCH Paul, NAUDY Carole*, COURANT Daniel*, PEROT Jean Claude** and GARCIA Jos6**.

Laboratoire de Radiotoxicologie et (*) Laboratoire de Neuropatholog!e Expdrimentale et Neurovirologie/DSV/DPTE, Commissariat h l'Energle Atomique, BP 12, 91680 - Bruy?~res-le-Chatel, France. (**) Laboratoire des Lasers ETCA-CREA, 94114 Arcueil, France.

Exposure to laser beams can induce small retinal lesions but these alterations as single spots have been poorly studied because their identification using standard histological methods is very time comsuming.The aim of this work was to develop an experimental procedure to study the evolution of single retinal lesions induced in rabbit after exposure to trains of picosecond laser pulses (200,000 pulses, 590 nm, 8.10"12s, IMHz). The retinal image diameter of the beam was 15-30 gm and 3 to 5 spots were performed on each retina which were distributed over about 1 cm 2 area to overcome lesion interactions. The experimental approach we have developed corresponded to 1) observation of 100 mm thick (100 gin) serial sections by DIC and scanning confocal laser microscopy to identify and to characterize lesions by fluorescence microscopy after different specific stainings, 2) epoxy resin embedding of lesion areas for further studies by light and standard electron microscopy. Animals were killed 4.5, 24, 48 and 96 hours after exposure. Retina were fixed overnight with 2% glutaraldehyde 2% paraformaldehyde in buffered solution (0.1 M cacodylate pH 7.3) after gradual dissection of the eye immersed in the fixative. The exposed area was cut, treated during at least 2 hours in the buffer containing I0 % saccharose and 10 % DMSO before freezing. Nuclei were stained with propidium iodide which allows to identify easily condensed DNA in apoptotic cells and a O-gal residues were stained by bandeiraea simplicifolia lectin labelled with F1TC which visualizes blood vessels and macrophages. Preliminary results show that about 20, 80 and 10 % homogeneously distributed apoptotic cells were confined to focal lesions in the outer nuclear layer after 4.5, 24 and 48 hours respectively. Macrophages were the only inflammatory cells in these lesions and the corresponding segment areas after 24 and 48 h. Only a few macrophages were encountered per lesion (<10). Electron microscopy allowed to characterize at 4.5 h pigmented epithelial cell alterations and to observe intralesional unphagocyted material up to 48 h. No lesion were detected "after 96 h suggesting a nearly complete repair.

UL'IRASTRUC/URE OF FAT STORING Cl~l I ,q IN THE RAT LUNG: AN ULTRATHIN SERIAL SECIION STUDY

Frans VAN MEIR. Pieter ILL. GYS and Dietrich W. SCHEUERMANN Laborc#ory of Cell Biology ~ d Histology, Detx~tment of Morphology, University of Antwerp (RUCA), B 2020 Antwerper~ Belgium

The blood-air barrier consists of a continuous epithelium lining the alve- olar space, endothelium lining the capillaries and interstitial cells in be- tween. In the interstitium fat storing cells are numerous in the prenatal and early postnatal rat lung. In adult animals we were able to induce the occurance of fat storing cells by adding vitamine A to their diet. There is nearly a tenfold increase in fat containing cells.

Electron microscopy of the blood-air barrier revealed significant changes in the morphology of the fat storing cells in the experimental rats, that received 90.000 - 120.000 I.E./day of retinolpalmitate for 12-20 days in their food. From two fat storing cells a series of respectively 76 and 138 ultrathin sections was studied.

It is clear that in hypervitaminosis A the number and size of the lipid droplets increased in the cytoplasma. The lipid droplets are densely pac- ked and seemed to be fused sometimes. The cell nucleus can get very indentated, while close contact between the nucleus and the lipid droplets is obvious. Serial sectioning reveals large cell extensions provided with large lipid droplets. These droplets have a large interlace area with the plasmamembrane.

Analognous to the cells of lto in the liver, the lipid droplets arise in number and size and the nucleus can be very defbrmed. From the fat storing cells in the lung it is known that they play a role in the perinatal lung development. In adult animals it still remains a matter of controversy if these cells can participate in repair processes and/or play a role in lung fibrosis.