Intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice

Abstract

Viral upper respiratory infections are the most common cause of clinical olfactory dysfunction, but the pathogenesis of dysosmia after viral infection is poorly understood. Biopsies of the olfactory mucosa in patients that complain of dysosmia after viral infection fall into two categories: one in which no olfactory epithelium is seen and another in which the epithelium is disordered and populated mainly by immature neurons. We have used intranasal inoculation with an olfactory bulb line variant of MHV to study the consequences of viral infection on peripheral olfactory structures. MHV OBLV has little direct effect on the olfactory epithelium, but causes extensive spongiotic degeneration and destruction of mitral cells and interneurons in the olfactory bulb such that the axonal projection from the bulb via the lateral olfactory tract is markedly reduced. Moreover, surviving mitral cells apparently remain disconnected from the sensory neuron input to the glomerular layer, judging from retrograde labeling studies using Dil. The damage to the bulb indirectly causes a persistent, long-term increase in the turnover of sensory neurons in the epithelium, i.e. the relative proportion of immature to mature sensory neurons and the rate of basal cell proliferation both increase. The changes that develop after inoculation with MHV OBLV closely resemble the disordering of the olfactory epithelium in some patient biopsies. Thus, damage to the olfactory nerve or bulb may contribute to a form of post-viral olfactory dysfunction and MHV OBLV is a useful model for studying the pathogenesis of this form of dysosmia.

Figure 1

Intranasal inoculation with MHV OBLV causes little damage to the epithelium during the acute phase of infection. (A—D) Age-matched control animal. The boxed area of the hematoxylin and eosin (H&E) stained section in (A) is shown in higher power in (B). (C, D) Comparable location in sections adjacent to (A) stained with anti-OMP and anti-GAP-43, respectively. (E—H)Ad libitum fed mouse inoculated intranasally with MHV OBLV 7 days prior to perfusion. Conventions as in (A)—(D). There are slightly fewer OMP+ neurons in the infected animal, but no increase in GAP-43+ cells, indicating that the epithelium is undamaged and unreactive (cf. Figure. 2). Arrowheads mark the basal lamina. Magnifications: (A, E), 40×; (B—D, F—H), 180×.

Figure 2

Occasional foci in the epithelium are more severely damaged after acute infection with MHV OBLV. The illustrated case was ad libitum fed at time of inoculation. (A, B) Anti-GAP-43 staining. Boxed area in (A) shown at higher power in (B). (C, D) Comparable location in sections adjacent to (A) that are stained with anti-OMP and H&E, respectively. Note that the epithelium is grossly intact judging from H&E stained material, but that mature, OMP+ sensory neurons have been lost and immature, GAP-43+ sensory neurons are increased as compared with the area of the epithelium illustrated in Figure 1. Arrowheads mark the basal lamina. Magnifications: (A), 38×; (B-D), 143×.

Figure 3

Intranasal inoculation with MHV OBLV causes widespread spongiotic degeneration in the OB during the acute phase of infection that reaches a maximum at 14 days after inoculation. (A-C) H&E stained sections of the bulb from mice surviving 7 and 14 days and an uninoculated control, respectively. All mice were ad libitum fed. Boxed areas in (A)-(C) are shown at higher magnification in (D-H), as indicated. (A, D) At 7 days note the spongiotic change in the outer EPL (epl), immediately deep to the GL (gl). (B, E, F) By 14 days there is frank separation of the deeper layers of the bulb from the GL and ONL due to obliteration of the EPL. There has also been some loss of granule cells as compared with controls (cf. H). (I) Photomicrograph from a comparable level of the bulb from a mouse that survived for 21 days after inoculation. The spongiotic change is resolving, but primary mitral cell dendrites are truncated and form swollen pale endings (arrowheads). igl, internal granule layer; mcl, mitral cell layer. Magnifications: (A-C), 16×; (D-H), 120×; (I), 260×.

Figure 4

The lateral olfactory tract undergoes spongiotic degeneration during the acute phase of infection and is reduced in cross-sectional area in the long-term, which suggests that mitral cell axons are lost from the tract as a consequence of infection. (A, B) Age-matched control. (C, D) Virally infected, ad libitum fed, 14 day post-infection. (E, F) 5 months post-infection. Boxed areas are shown at higher magnification, as indicated. lot, LOT; I, layer I of piriform cortex; II, layer II of piriform cortex; III, layer III of piriform cortex. Magnifications: (A, C, E), 85×; (B, D, F), 170×.

Figure 5

Spread of MHV OBLV to more central structures. (A) anti-MHV-immunoreactive pyramidal neurons in layer II of piriform cortex. (B) Focus of degeneration and mononuclear infiltrate in the caudate nucleus (arrowheads). Ad libitum fed mouse. Magnifications: (A), 180× ; (B), 270 ×.

Figure 6

The structure of the OB remains distorted at long survival times after infection. In addition, the projection from the epithelium onto the bulb consists of a larger than normal population of newly innervating axons in infected animals as compared with controls. (A, B, E) Age-matched control. (C, D, F) Mouse inoculated with MHV OBLV and then killed 5 months after infection, food-restricted at time of inoculation. (A, C) Staining with anti-OMP. (B, D, E, F) Staining of adjacent section with anti-GAP-43. Note the marked reduction in thickness of the EPL in the lesioned animals (D, F versus B, E), the presence of OMP-stained sensory axons in the GL of the infected animal (C) and the increase in GAP-43 stained sensory axons in the ONL and GL of the infected animal (D, F versus B, E). Abbreviations as in Figure 3. Magnifications: (A-D), 23×; (E, F), 117×.

Figure 7

Mitral cells are lost as a consequence of infection. (A, B) Age-matched control. (C, D) Mouse killed 5 months after inoculation, ad libitum fed at time of inoculation. Note the lack of a defined mitral cell layer (A versus C) and loss of mitral cells (arrows in B, D) after lesion. Abbreviations as in Figure 3. Magnifications: (A, C), 108×; (B, D), 315×.

Figure 11

At long survival times after inoculation the number of proliferating basal cells is increased. Furthermore, the degree of damage to the EPL correlates with the increase in the index of basal cell proliferation in MHV OBLV-infected mice (inoculated when food-restricted) that survived for 5 months after inoculation. (A) Age-matched control, injected i.p with [³H]thymidine 2 h before perfusion. (B) Mouse killed 5 months post-inoculation, ad libitum fed at the time of inoculation, injected i.p. with [³H]thymidine 2 h before perfusion. Arrows indicate [³H]thymidine labeled basal cells. (C) Area of the epithelium illustrated in (A); (B) is indicated by the open arrow. (D) Plot of labeling index versus area of the EPL for each of the experimental (squares) and control (circles) animals. As discussed in the text, one of the three mice in the inoculated group was affected only minimally by the virus in terms of clinical symptomatology and pathological changes to the bulb (curved arrow); presumably the dose of virus was insufficient to produce significant disease in that particular case for technical reasons, most likely leakage of the inoculum. The overall correlation between these two measures is significant across all of the animals. The outlier may be excluded from the experimental data set on the grounds that infusion of virus was clinically ineffective in that case. With this exclusion the difference in labeling index between the two groups is highly significant. Magnifications: (A, B), 270×.

Figure 8

Granule cell number partially recovers with time after lesion. (A) H&E stained section of olfactory bulb from a representative mouse killed 1 month after inoculation, food-restricted at time of inoculation. Note the paucity of granule cells relative to control (cf. Figure 3C). (B) H&E stained section of olfactory bulb from a representative mouse killed 5 months after inoculation, also food-restricted at time of inoculation. Note the apparent increase in the number of granule cells that has ensued in the prior 4 months. (C) Counts of granule cells confirm the visual impression that granule cell numbers increase between 1 and 5 months after infection. Average number of cells per level, determined bilaterally at five equally spaced levels through the antero-posterior extent of the bulb; comparable levels were counted in each case. Each triangle designates an individual experimental case. The horizontal line indicates the group mean and the error bar represents the SEM. Magnifications: (A, B), 27×.

Figure 9

Dil labeling demonstrates that the GL (gl) is largely disconnected from the deeper layers of the bulb in the virally infected mice. (A-D) Age-matched control. (E-H) Mouse killed 5 months after inoculation, ad libitum fed at time of inoculation. Boxed areas shown at higher magnification as indicated. At the level of the anterior olfactory nucleus (AON) retrograde labeling is prominent in the lateral olfactory tract and in the neurons of the AON of the control animal (A) but less so in the MHV OBLV-infected animal (E), despite roughly equivalent injection areas in the piriform cortex in the two animals. (B, C) In the control OB label ends at the boundary between the GL (gl) and the ONL (onl), i.e. the GL is labeled heavily and the ONL is unlabeled. (F, G) In contrast, in the infected mouse label ends at the boundary between the EPL (epl) and GL around much of the circumference of the bulb, i.e. the amount of label in the GL is roughly comparable to that in the ONL. An area of the ventrolateral bulb where some labeling extends into the GL of the inoculated mouse is indicated by the arrows in (F). The short vertical lines mark the boundaries between layers. (D, H) Transmitted light images of sections illustrated in (B), (F), respectively. Abbreviations as in Figure 3. Magnifications: (A, B, D-F, H, 18×; (C, G), 38×.

Figure 10

At long survival times after inoculation the number of immature sensory neurons is increased and the number of mature sensory neurons is decreased by comparison with normal. (A-C) Age-matched control. (D-F) Mouse killed 5 months after inoculation, food-restricted at time of inoculation. (A, B, D, E) Anti-GAP-43 immunostaining. Note that the number of GAP-43+ neurons is increased throughout much of the circumference of the epithelium of the lesioned mouse. (C, F) Anti-OMP immunostaining of the same area of the epithelium from sections adjacent to (A and B) and (D and E), respectively. The number of OMP+ neurons is correspondingly reduced in the lesioned animal. Magnifications: (A, D), 57×; (B, C, E, F), 150×.