Manganese uptake and distribution in the brain after methyl bromide-induced lesions in the olfactory epithelia

Abstract

Manganese (Mn) is an essential nutrient with potential neurotoxic effects. Mn deposited in the nose is apparently transported to the brain through anterograde axonal transport, bypassing the blood-brain barrier. However, the role of the olfactory epithelial cells in Mn transport from the nasal cavity to the blood and brain is not well understood. We utilized the methyl bromide (MeBr) lesion model wherein the olfactory epithelium fully regenerates in a time-dependent and cell type-specific manner over the course of 6-8 weeks postinjury. We instilled (54)MnCl(2) intranasally at different recovery periods to study the role of specific olfactory epithelial cell types in Mn transport. (54)MnCl(2) was instilled at 2, 4, 7, 21, and 56 days post-MeBr treatment. (54)Mn concentrations in the blood were measured over the first 4-h period and in the brain and other tissues at 7 days postinstillation. Age-matched control rats were similarly studied at 2 and 56 days. Blood and tissue (54)Mn levels were reduced initially but returned to control values by day 7 post-MeBr exposure, coinciding with the reestablishment of sustentacular cells. Brain (54)Mn levels also decreased but returned to control levels only by 21 days, the period near the completion of neuronal regeneration/bulbar reinnervation. Our data show that Mn transport to the blood and brain temporally correlated with olfactory epithelial regeneration post-MeBr injury. We conclude that (1) sustentacular cells are necessary for Mn transport to the blood and (2) intact axonal projections are required for Mn transport from the nasal cavity to the olfactory bulb and brain.

FIG. 1.

Experimental design of MeBr exposure protocol. Male Sprague-Dawley Germfree rats, 9 weeks old (287 ± 5.8 g), were obtained from Taconic Farms. After 3–4 days of acclimatization in the animal facilities, rats were transported to the Department of Anatomy and Cellular Biology, Tufts University School of Medicine, and subjected to MeBr exposure (Day 0). Rats were returned to HSPH animal facility at the end of exposure and were sequentially studied at 2, 4, 7, 21, and 56 days after MeBr exposure when rats were instilled intranasally with ⁵⁴MnCl₂ (Perkin Elmer/NEN) at 30 μCi/kg and a volume of 0.1 ml/kg body weight (four rats per group). All instilled rats were analyzed 7 days later for brain and other tissue concentration of ⁵⁴Mn. *, At these times, separate cohorts of untreated age-matched control rats were also studied.

FIG. 2.

Blood (A) and brain (B) concentrations of ⁵⁴Mn after a single intranasal instillation or iv injection of ⁵⁴MnCl₂ in normal CD rats. Rats were intranasally instilled or iv injected with 7.5 μCi/kg ⁵⁴MnCl₂ and analyzed at 4 h (open bars) or 72 h (closed bars) postdosing. Each point is a mean ± SE of four rats. The ⁵⁴Mn brain concentration was initially higher after iv than after intranasal instillation at 4 h (⁺). However, from 4 to 72 h, the brain levels significantly increased after intranasal instillation but not after iv dosing (*). Moreover, the ⁵⁴Mn brain levels at 72 h were significantly higher after intranasal instillation than after iv administration (⁺⁺). (*^,+,++p < 0.05, Student's t-test)

FIG. 3.

⁵⁴Mn blood concentrations in control and in MeBr-treated rats at 1, 2, and 4 h after ⁵⁴Mn intranasal instillation. Rats were exposed to MeBr and allowed to recover 2, 4, 7, 21, or 56 days prior to ⁵⁴MnCl₂ instillation. (A) Control rats were not exposed to MeBr. Blood ⁵⁴Mn levels are nanocurie per gram. Each point is a mean ± SE of four rats. Multivariate ANOVA showed that the different groups of MeBr-exposed rats were significantly different from the 9-week-old controls. Significant decrease over time was also seen in all rat groups (*p < 0.05). ⁵⁴Mn blood concentrations in 56-day-recovered MeBr-treated rats and age-matched controls (17 weeks old) were not different (B).

FIG. 4.

Brain and nonneuronal tissue uptake of ⁵⁴Mn. Blood (A), kidney (B), and liver (C) concentrations of ⁵⁴Mn were significantly decreased at 4 days post-MeBr treatment but returned to normal by day 7 of recovery. In contrast, the total brain (D) concentrations were significantly decreased at 2, 4, and 7 days and returned to control values only from day 21 post-MeBr treatment.

FIG. 5.

Comparison of intranasally instilled versus iv-injected ⁵⁴MnCl₂ in MeBr-exposed rats. Shown are brain tissue concentrations of ⁵⁴Mn after iv injection (A) or intranasal instillation (B) of ⁵⁴MnCl₂ in MeBr-exposed (closed bars) or age-matched untreated controls (open bars) 2 days after MeBr exposure. The retained ⁵⁴Mn in all dissected brain regions was significantly decreased in MeBr-exposed rats intranasally instilled with the radioisotope. In contrast, only the olfactory bulb had significantly reduced ⁵⁴Mn in MeBr-exposed iv-injected rats. *p < 0.05. (Basal ganglia⁺, these data excluded the substantia nigra).

FIG. 6.

⁵⁴Mn levels in dissected brain regions of intranasally instilled MeBr-treated rats. Rats were instilled with ⁵⁴MnCl₂ at specific time point post-MeBr treatment (closed circles): 2, 4, 7, 21, or 56 days. Two groups of weight-matched control rats were not exposed to MeBr and were studied at the beginning and at the end of the 56-day MeBr postexposure recovery period (open circles). Seven days after intranasal instillation of ⁵⁴MnCl₂, rats were humanely killed and brains were collected for microdissection. Brain tissue ⁵⁴Mn levels are expressed as nanocurie per gram. Figure 6A depicts phases of OE regeneration along the time line of our experiment. In all the brain regions, there was a significant reduction in ⁵⁴Mn concentration at 2, 4, and 7 days post-MeBr exposure (B–H). The values for all brain regions were back to control levels at 21 days postexposure. The return to control levels correlates well with the end of the intermediate phase of OE regeneration (A). Each point is a mean ± SE of four rats. *p < 0.05. (Basal ganglia⁺, these data excluded the substantia nigra).