Nitric oxide is protective in listeric meningoencephalitis of rats

Abstract

The bacterium Listeria monocytogenes causes meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation with L. monocytogenes on survival and activity were noted; 10(4) L. monocytogenes organisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO.) production. Treatment with the spin-trapping agent alpha-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO. contribute to Listeria growth control.

FIG. 1

Survival (A) and activity (B) profiles of rats inoculated intracisternally with various doses of L. monocytogenes. Data were compiled from three experiments; each dose group contained four or more animals. Symbols refer to doses of L. monocytogenes as follows: closed squares, 10³ CFU; open triangles, 10⁴ CFU; open circles, 10⁵ CFU; open diamonds, 10⁶ CFU.

FIG. 2

Growth kinetics of and tissue infiltration by L. monocytogenes in the brains of intracisternally infected rats. Tissues were analyzed at various times after infection with 10⁴ CFU of L. monocytogenes. (A) Scores taken in a blind fashion in meninges (closed symbols) and in plexus and periventricular regions (open symbols) were averaged for individual animals as described in Materials and Methods. Each group consisted of a minimum of five rats. The horizontal lines indicate the medians of the groups (B) CFU of L. monocytogenes in spleen (hatched dots), liver (open dots), and cerebellum (black dots) tissues. For animals with no detectable bacteria, the threshold of detection was entered. The detection threshold is indicated in the graph by the dotted line.

FIG. 3

Rat brain sections showing areas of inflammation due to infection with L. monocytogenes around the third ventricle. They were stained for bacteria (red, panels A to D), iNOS (brown, panels A to E), or NT (brown, panel F). L indicates the lumen of the ventricle. TD indicates tissue destruction due to massive inflammation. Arrows point to intracellular bacteria. (A) Ependymal tissue at day 3. (B) Subependymal tissue at day 5. (C) Sequestration of bacteria at day 5. (D) Clearing of infection at day 7. (E) Overview of the third ventricle at day 3 after infection showing staining for iNOS; insert, iNOS-expressing Mφ. (F) Section adjacent to E stained for NT, insert, NT granules in Mφ. Magnification, ×400 (A to D), ×100 (E and F), and ×3,000 (inserts of E and F). The bars in E and F represent 1 mm.

FIG. 4

Kinetics and tissue distribution of iNOS (A) and NT (B) in the brains of intracisternally infected rats. Tissues were analyzed at days 3, 5, and 7 postinfection with 10⁴ CFU of L. monocytogenes. Scores were determined in meninges (filled triangles) and in the plexus and periventricular regions (open triangles) as described in Materials and Methods. The animals analyzed here were the same as those used for Fig. 2. The horizontal lines indicate the medians of the groups.

FIG. 5

Survival of rats infected with L. monocytogenes and treated with either PBN (open circles) or L-NIL (open triangles) or left untreated (closed squares). The graph incorporates several experiments. Each group represents at least 13 animals.

FIG. 6

L. monocytogenes growth in organs of animals treated with a selective iNOS inhibitor, L-NIL. (A) L. monocytogenes score at day 2 as determined in L-NIL-treated (open triangles) or mock-treated animals (closed triangles). (B) CFU in spleen, liver, and cerebellum tissues at day 2 after treatment with L-NIL (hatched dots) or mock treatment (open dots). The graph accounts for 15 animals per group, infected at different days. The difference between the control group and the L-NIL-treated group is significant (P < 0.01) for the cerebellum. The dotted line indicates the threshold of detection. Horizontal bars indicate the medians in each panel.

FIG. 7

L. monocytogenes growth in organs of animals treated with PBN. (A) L. monocytogenes scores at day 2 as determined in PBN-treated (open triangles) or mock-treated animals (closed triangles). (B) CFU in cerebellum, spleen, and liver tissues at day 2 after treatment with PBN (hatched dots) or mock treatment (open dots). The graph accounts for 13 animals per group, infected at different days. The differences in the results of control and PBN-treated tissues are significant (P < 0.001) for all groups. The dotted line indicates the threshold of detection. Horizontal bars represent medians of the groups in each panel.