Central Nervous System Tuberculosis in a Murine Model: Neurotropic Strains or a New Pathway of Infection?

Abstract

Tuberculosis (TB) of the central nervous system (CNS) is a lethal and incapacitating disease. Several studies have been performed to understand the mechanism of bacterial arrival to CNS, however, it remains unclear. Although the interaction of the host, the pathogen, and the environment trigger the course of the disease, in TB the characteristics of these factors seem to be more relevant in the genesis of the clinical features of each patient. We previously tested three mycobacterial clinical isolates with distinctive genotypes obtained from the cerebrospinal fluid of patients with meningeal TB and showed that these strains disseminated extensively to the brain after intratracheal inoculation and pulmonary infection in BALB/c mice. In this present study, BALB/c mice were infected through the intranasal route. One of these strains reaches the olfactory bulb at the early stage of the infection and infects the brain before the lungs, but the histological study of the nasal mucosa did not show any alteration. This observation suggests that some mycobacteria strains can arrive directly at the brain, apparently toward the olfactory nerve after infecting the nasal mucosa, and guides us to study in more detail during mycobacteria infection the nasal mucosa, the associated connective tissue, and nervous structures of the cribriform plate, which connect the nasal cavity with the olfactory bulb.

Keywords: central nervous system tuberculosis; experimental tuberculosis; neurotropism; tuberculosis animal model.

Figure 1

(A) Survival Kaplan–Meier plot; (B) Lung bacillary load survival curves were constructed with 20 infected mice per bacterial strain and analyzed with Kaplan–Meier plots and the Log Rank test (Mantel-Cox). Data are the mean ± SD of measurements from three mice per time point in three different experiments. Asterisks represent statistical significance (p < 0.05) when compared with H37Rv strain infection.

Figure 2

Representative BALB/c mice lung histopathology after infection with M. tuberculosis strain 209. (A) Tissue without histopathological changes at 1 dpi. (B) Granuloma, delimited by the dashed line, surrounded by chronic inflammatory infiltrate at 14 dpi, (C) Focal pneumonia and chronic inflammatory infiltrate around blood vessels (arrow) and airways at 21 dpi and minimal intralveolar inflammation. (D) Extensive pneumonia at 60 dpi. Micrographs (A,C) are at 100× magnification stained with H&E, and micrographs (B,D) are at 400× magnification stained with H&E.

Figure 3

Representative BALB/c mice lung histopathology after infection with M. tuberculosis H37Rv. (A) Tissue without histopathological changes at 1 dpi. (B) Well-consolidated subpleural granuloma, the rest of parenchyma without pneumonia (*) at 14 dpi. (C) Granuloma surrounded by an inflammatory infiltrate at 120 dpi, (D) Parenchyma with pneumonia, consolidation and acid fast bacilli (AFB) aggregate dyed in fuchsia color (arrows) at 120 dpi, (Micrographs (A,C) at 400 magnification, micrograph (B) at 100 magnifications, stained with H&E, micrograph (D) at 400 magnification and stained with ZN, granulomas delimited by the dashed line).

Figure 4

Bacillary load in BALB/c mice infected by an intranasal injection of M. tuberculosis isolates of the olfactory bulb (A) and brainstem (B). Reference strain H37Rv was used as a control. Data are the mean ± SD of measurements from three mice per time point in three different experiments. (C) Encephalon bacillary load in BALB/c mice infected by intranasal injection with M. tuberculosis isolates. Reference strain H37Rv was used as a control. Asterisks represent statistical significance (p < 0.05) when compared with H37Rv strain infection.

Figure 5

Representative BALB/c mice encephalon histology after infection with M. tuberculosis strain 209. (A) Olfactory bulb, (B) Brain (C) Cerebellum, and (D) Brainstem. Abnormal Morphological changes were not detected in any section of these anatomic regions. (All micrographs at 100× magnification and stained with H&E at 120 dpi.

Figure 6

Representative BALB/c mice olfactory area histology after infection with M. tuberculosis strain 209. (A) Detailed view of the olfactory bulb showing intact tissue without inflammatory or infectious alterations. (B) Close examination of the olfactory mucosa, ethmoid bone, and olfactory bulb, none of the structures show histological damage or inflammation that indicate an infectious process. (Micrographs At 100× magnification stained with H&E, micrograph B at 400× magnification stained with H&E at 120 dpi.

Figure 7

Bacillary load in BALB/c mice infected by an intranasal injection of M. tuberculosis isolates of the kidney (A), liver (B), and spleen (C). Reference strain H37Rv was used as control. Data are the mean SD of determinations from three mice per time point in three different experiments. Asterisks represent statistical significance (p < 0.05).

Figure 8

Classical Pathway. Stage 1a. Initial Infection: Mice inhale droplets containing mycobacterium tuberculosis (Mtb). Stage 2a. Pulmonary Manifestation: In the lungs, Mtb gets engulfed by alveolar macrophages. Within these cells, the bacterium can multiply or remain dormant. The body’s immune response tries to contain the infection, leading to the formation of Ghon’s foci, which are small nodules or lesions. Stage 3a. Lymphohematogenous Dissemination: If Ghon’s foci rupture, free mycobacteria can enter the bloodstream. Stage 4a. “The Trojan Horse Mechanism”: This spread causes Mtb to colonize the vascular endothelium of arachnoid mater vessels, leading to damage in the brain–blood barrier (BBB). Infected macrophages (carrying the bacteria) can cross the vascular endothelium into the CNS. This mechanism is likened to the famous “Trojan horse” myth because the bacteria, hidden inside the macrophages, gain entry to otherwise protected sites. Stage 5a. Cerebral infection. Alternative hypothesis. Stage 1b. Early CNS Invasion Post Intranasal Inoculation: After introducing Mtb through the nasal pathway, early CNS invasion can be observed from day 1. Stage 2b. Certain strains of Mtb might not require pulmonary infection to establish a CNS infection. These strains could potentially bypass the lungs and directly colonize the CNS. While mycobacteria are detectable in the lungs, classical signs of CNS invasion, such as Ghon’s foci, are absent at early stages. Stage 3b. Olfactory Bulb Colonization: Mtb is found in the olfactory bulb throughout the course of infection. This suggests the possibility of CNS infection initiating from peripheral nerves or lymphatic vessels in the nasal mucosa, allowing Mtb to reach the olfactory bulb and establish an infection. Stage 4b. Mtb’s progression unfolds via two distinct mechanisms: firstly, through the inflammatory response, where activated immune cells strive to contain the pathogen; and secondly, via neural infiltration, where Schwann cells facilitate the bacterium’s ascendancy, echoing the intricate cellular interplay in infection dynamics. Stage 5b. Absence of cerebral damage: No significant histological changes are observed in the brain of infected mice, homologous to mycobacterium leprae behavior in the human nasal mucosa.