Olfactory and trigeminal routes of HSV-1 CNS infection with regional microglial heterogeneity

Abstract

Herpes simplex virus type 1 (HSV-1) primarily targets the oral and nasal epithelia before establishing latency in the trigeminal ganglion (TG) and other peripheral ganglia. HSV-1 can also infect and become latent in the central nervous system (CNS) independent of latency in the TGs. Recent studies suggest entry to the CNS via two distinct routes: the TG-brainstem connection and olfactory nerve; however, to date, there is no characterization of brain regions targeted during HSV-1 primary infection. Furthermore, the immune response by microglia may also contribute to the heterogeneity between different brain regions. However, the response to HSV-1 by microglia has not been characterized in a region-specific manner. This study investigated the time course of HSV-1 spread within the olfactory epithelium (OE) and CNS following intranasal inoculation and the corresponding macrophage/microglial response in a C57BL/6 mouse model. We found an apical to basal spread of HSV-1 within the OE and underlying tissue accompanied by an inflammatory response of macrophages. OE infection was followed by infection of a small subset of brain regions targeted by the TG in the brainstem and other cranial nerve nuclei, including the vagus and hypoglossal nerve. Furthermore, other brain regions were positive for HSV-1 antigens, such as the locus coeruleus (LC), raphe nucleus (RaN), and hypothalamus while sparing the hippocampus and cortex. Within each brain region, microglia activation also varied widely. These findings provide critical insights into the region-specific dissemination of HSV-1 within the CNS, elucidating potential mechanisms linking viral infection to neurological and neurodegenerative diseases.IMPORTANCEThis study shows how herpes simplex virus type 1 (HSV-1) spreads within the brain after infecting the nasal passages. Our data reveal the distinct pattern of HSV-1 through the brain during a non-encephalitic infection. Furthermore, microglial activation was also temporally and spatially specific, with some regions of the brain having sustained microglial activation even in the absence of viral antigens. Previous reports have identified specific brain regions found to be positive for HSV-1 infection; however, to date, there has not been a concise investigation of the anatomical spread of HSV-1 and the brain regions consistently vulnerable to viral entry and spread. Understanding these region-specific differences in infection and immune response is crucial because it links HSV-1 infection to potential triggers for neurological and neurodegenerative diseases.

Keywords: Alzheimer's disease; HSV-1; microglia; olfactory.

Fig 1

Experimental design of intranasal inoculation of HSV-1. (A) C57BL/6 mice were intranasally inoculated with HSV-1 (McKrae strain, 10⁶). (B, C) Representation of regions of the brain is positive for HSV-1 antigen and/or microglial activation, including OE, OB, diencephalon (Di), and brainstem (BS) subnuclei. (D) Timeline of infection and tissue harvest; for OE, tissue was harvested at 7 hours post-inoculation and 3 days post-inoculation with HSV-1. (E) For the survival curve, C57BL6 mice were intranasally inoculated with HSV-1 McKrae strain (n = 14), and Mock (n = 5) was followed out for 14 days post-inoculation. By 8 days post-inoculation, 2/14 HSV-1-infected animals had died but were not significantly different from controls (P = 0.38, by log-rank test).

Fig 2

Distribution of HSV-1 antigen throughout the nasal cavity. (A) Diagram depicting mouse nasal structure and the specific areas where the olfactory epithelium was analyzed for immunohistochemistry. In anterior sections (i), the tissue lining the nasal cavity is made of respiratory epithelium (colored in magenta), whereas it is made of olfactory epithelium in posterior sections (ii, iii, and iv, colored in orange). Steno’s gland (green), nasal-associated lymphoid tissue (blue), and olfactory bulb (yellow) are also represented on the diagrams to help visualize the nasal anatomy. (B, C, D) Representative images of infected tissue in the nasal cavity, 7 hours, and 3 days post-inoculation with HSV-1. Debris in the lumen of the nasal cavity are indicated with an asterisk. Arrowheads point to infection of the apical layer of the olfactory epithelium. The scale bar is 100 µm. (E) Quantification of the surface of infected tissue across entire sections of the nasal cavity. The surface of HSV-1-positive tissue was calculated as a percentage of the total length of respiratory (RE) and olfactory epithelium (OE). 4 sections per animal were used for the quantification, one from each area described in (A). n = 2 and n = 3 animals for 7 h and 3 DPI, respectively. Data are represented as mean ± sem.

Fig 3

HSV-1 infection of the OE is associated with immune cell infiltration and tissue disruption. (A) A portion of OE from a mock-infected animal showing IBA1+ cells in the basal side of the OE. (B) Portion of OE from an infected animal (3 DPI) showing an infection spot in the OE (magenta) and infiltration of IBA1+ cells throughout the OE (asterisk). (C) Same area as in (B) but labeled with OMP (olfactory neurons marker). The left side (arrowheads) shows a non-infected area of OE with preserved olfactory neurons. The infection spot on the right side is associated with complete tissue disruption (asterisk). Tissue debris is also seen in the lumen of the nasal cavity (delimited with a dotted line). (D) High-magnification image of the cell framed in (C) showing multiple nuclei in the same infected cell envelope.

Fig 4

Microglia activation in the OB in the absence of HSV-1 infection. (A) Mouse brain atlas image of the olfactory bulb (OB; Bregma = ~7.32–6.56 mm) and corresponding representative histological image of a C56BL6 infected with HSV-1 at 10 days post-inoculation, stained for HSV-1 (magenta), IBA1 (green), and DAPI (blue). (B) Representative histological images taken from the olfactory bulb of mice inoculated with HSV-1 (Mckrae, 10⁶) at 1, 3, 7, and 10 days post-inoculation. (C) Schematic and representative morphology of microglial activation states based on morphology. A score of 1 = ramified microglia (small cell body and long cellular processes, 2 = hyper-ramified (thicker and more branching projections), 3 = bushy (fuzzy appearance, larger cell body, thicker projections), 4 = transitional (between busy and amoeboid, thick small projections), 5 = amoeboid = no ramified projections. (D) Quantification from each animal in D for microglia activation for 1 (n = 188), 3 (n = 144), 7(n = 80), and 10(n = 237) days post-inoculation. Each point is individual microglia analyzed for activation state, P < 0.001, Kruskal-Wallis test.

Fig 5

Histological assessment of microglial activation within cranial nerve brainstem nuclei. (A) A representative histological image of a mouse brain at 7 days post-inoculation taken from the brainstem nuclei (~−4.04 mm: −3.52 mm from Bregma) cranial nerve XII nuclei (Hypoglossal nerve nuclei, XIIN). Quantification of microglia activation in XIIN at day 1 (n = 20), 3 (n = 30), day 7 (n = 23), and 10 (n = 25) post-inoculation. (B) The nucleus of the solitary tract (NoST). Quantification of microglia activation in NoST at day 1 (n = 90), 3 (n = 44), day 7 (n = 44), and 10 (n = 123) post-inoculation. (C)Cranial nerve X (vagal nerve nuclei, DMX). Quantification of microglia activation in DMX at day 1 (n = 9), 3 (n = 32), day 7 (n = 39), and 10 (n = 34) post-inoculation. (D) Area postrema (AP). Quantification of microglia activation in AP at day 1 (n = 38), 3 (n = 53), day 7 (n = 88), and 10 (n = 56) post-inoculation. (E) Spinal trigeminal nerve nuclei (SPVC). Quantification of microglia activation in SPVC at day 1 (n = 54), 3 (n = 66), day 7 (n = 92), and 10 (n = 140) post-inoculation. Histological analysis of HSV-1 glycoproteins (magenta), IBA1+ microglia (green), and nuclear stain DAPI. Each point is individual microglia analyzed for activation state, P < 0.001, Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: XIIN, hypoglossal nucleus; DMX, vagus nerve nucleus, dorsal motor; SPVC, spinal trigeminal nucleus; NoST, nucleus of the solitary tract; AP, area postrema. Each point is individual microglia analyzed for activation state, P < 0.001, Kruskal-Wallis test.

Fig 6

Histological assessment of microglia activation within neuromodulatory brainstem nuclei. (A) A representative histological image of a mouse brain at 7 days post-inoculation was taken from the brainstem nuclei (~−3.28 mm: −1.72 mm from Bregma) consisting of serotonergic centers raphe nucleus (RaN). Quantification from each region of microglia activation in RaN at day 1 (n = 123), 3 (n = 72), day 7 (n = 27), and 10 (n = 132) post-inoculation. (B) A representative histological image of a mouse brain at 7 days post-inoculation was taken from the brainstem nuclei (~−3.28 mm: −1.72 mm from Bregma) consisting of noradrenergic center of the locus coeruleus (LC). Quantification from each region of microglia activation in LC at day 1 (n = 54), 3 (n = 32), day 7 (n = 34), and 10 (n = 64) post-inoculation. Each point is individual microglia analyzed for activation state, P < 0.001, Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations; RaN raphe nucleus; LC, locus coeruleus.

Fig 7

Histological assessment of microglia activation within the diencephalon sub-nuclei. (A) A representative histological image of a mouse brain at 7 days post-inoculation was taken through the diencephalon (~−1.52 mm: 3.08 mm from Bregma) consisting of hypothalamic subnuclei, lateral hypothalamus (LH). Quantification of microglia activation in LH at day 1 (n = 136), 3 (n = 86), day 7 (n = 36), and 10 (n = 150) post-inoculation. (B) A representative histological image of dorsomedial hypothalamus (DMH). Quantification of microglia activation in DMH at day 1 (n = 124), 3 (n = 45), day 7 (n = 49), and 10 (n = 111) post-inoculation. (C) A representative histological image of the paraventricular nucleus (PVN). Quantification of microglia activation in PVN at day 1 (n = 45), 3 (n = 10), day 7 (n = 18), and 10 (n = 66) post-inoculation. (D) A representative histological image in the hippocampus (Hip). Quantification of microglia activation in Hip at day 1 (n = 57), 3 (n = 41), day 7 (n = 5), and 10 (n = 39) post-inoculation. Each point is individual microglia analyzed for activation state, P < 0.001, Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations; LH, lateral hypothalamus; DMH, dorsomedial hypothalamus; PVN, paraventricular nucleus; Hip, hippocampus.

Fig 8

Comparison of microglial activation states within distinct brain regions over the course of HSV-1 infection. (A) Representation of HSV-1 antigen presence over time between each brain region. Regions that were negative (gray) or positive (red) for HSV-1 antigen are represented for each brain region. (B) Microglial activation states from all the above brain regions were collated and reported as an intensity measure over the course of infection (1, 3, 7, and 10 days post-inoculation). Abbreviations: OB, olfactory bulb; XIIN, hypoglossal nucleus; DMX, vagus nerve nucleus, dorsal motor; SPVC, spinal trigeminal nucleus; NoST, nucleus of the solitary tract; AP, area postrema, RaN, raphe nucleus; LC, locus coeruleus, LH, lateral hypothalamus; DMH, dorsomedial hypothalamus; PVN, paraventricular nucleus; HIP, hippocampus; Gi, gigantocellular medullary reticular nucleus; MRNv, medullary nucleus-ventral; MVN, medial vestibular nucleus; LRN, lateral reticular nucleus; IRTA, intermediate reticular nucleus.