Using the olfactory system as an in vivo model to study traumatic brain injury and repair

Abstract

Loss of olfactory function is an early indicator of traumatic brain injury (TBI). The regenerative capacity and well-defined neural maps of the mammalian olfactory system enable investigations into the degeneration and recovery of neural circuits after injury. Here, we introduce a unique olfactory-based model of TBI that reproduces many hallmarks associated with human brain trauma. We performed a unilateral penetrating impact to the mouse olfactory bulb and observed a significant loss of olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) ipsilateral to the injury, but not contralateral. By comparison, we detected the injury markers p75(NTR), β-APP, and activated caspase-3 in both the ipsi- and contralateral OE. In the olfactory bulb (OB), we observed a graded cell loss, with ipsilateral showing a greater reduction than contralateral and both significantly less than sham. Similar to OE, injury markers in the OB were primarily detected on the ipsilateral side, but also observed contralaterally. Behavioral experiments measured 4 days after impact also demonstrated loss of olfactory function, yet following a 30-day recovery period, we observed a significant improvement in olfactory function and partial recovery of olfactory circuitry, despite the persistence of TBI markers. Interestingly, by using the M71-IRES-tauLacZ reporter line to track OSN organization, we further determined that inducing neural activity during the recovery period with intense odor conditioning did not enhance the recovery process. Together, these data establish the mouse olfactory system as a new model to study TBI, serving as a platform to understand neural disruption and the potential for circuit restoration.

Keywords: APP; apoptosis; biomarker; circuit repair; mouse model; olfactory system; p75NTR; regeneration; traumatic brain injury.

FIG. 1.

Schematic of experimental paradigm and injury parameters. (A) Dorsal view of a mouse head illustrating the location of the impact site over the olfactory bulb with associated coordinates and impact details. (B) Sagittal view of impact site with schematic of olfactory circuitry. Olfactory sensory neurons that express the same odorant receptor (depicted as purple or blue) in the olfactory epithelium project axons that converge onto the same location in the olfactory bulb to produce the glomerular olfactory map. Output neurons receive the olfactory information and transmit their signal to the piriform cortex. Impact to the dorsal bulb disrupts olfactory function and the stereotyped anatomical circuitry. OBI, olfactory bulb impact. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Olfactory sensory neuron loss after injury. (A) Cartoon depicting cellular organization in the olfactory epithelium (OE) and highlighting location of the mature olfactory sensory neurons (OSNs; OMP⁺) and the immature OSNs (growth-associated protein 43; GAP-43⁺). (B) Representative images of sham (top), contralateral (middle), and ipsilateral (bottom) OE in OMP-GFP mice. Sections used in quantification were taken from the dorsal septal region depicted by the red boxed region in (A). (C) Histogram comparing the number of cells per 250-micron region of the dorsal septum in injured and noninjured animals: sham/ipsilateral side (p<0.00008) and ipsilateral side/contralateral side (p<0.0000003). GFP, green fluorescent protein. Color image is available online at www.liebertpub.com/neu

FIG. 3.

Markers of traumatic brain injury (TBI) in the olfactory epithelium 4 days post-OBI (olfactory bulb impact). Representative images from sham, contralateral, and ipsilateral olfactory epithelium (OE) are shown from left to right. p75^NTR (A), beta amyloid precursor protein (β-APP) (B), cleaved caspase-3 (C), and growth-associated protein 43 (GAP-43) (D) show a differential change in TBI markers between the ipsi- and contralateral epithelium. Color image is available online at www.liebertpub.com/neu

FIG. 4.

Bilateral loss of olfactory bulb (OB) neurons after injury. (A) Representative images at the core injury site from sham, contralateral, and ipsilateral OB are shown from left to right with cartoon illustrating relationship of impact site and sampled regions. (B) Representative images at the penumbra from sham, contralateral, and ipsilateral OB are shown from left to right with cartoon illustrating sampled regions. (C) Graphs showing measurements of: 1=outer boundary of the glomerular layer (GL); 2=inner boundary of the GL and outer boundary of the external plexiform layer (EPL); 3=inner boundary of the EPL. Histograms showing the area measured adjacent to the impact (500–1000 microns ventral to the dorsal tip of the OB; top panels) and ventral to the impact (1000–1250 microns ventral to the dorsal tip of the OB; bottom panels) in the GL and EPL layers. (D) Histogram showing the area measured at the penumbra (anterior to the impact site) for the whole OB (1), GL (1–2), EPL (2–3), and granule cell layer (GCL) (3). Color image is available online at www.liebertpub.com/neu

FIG. 5.

Markers of traumatic brain injury (TBI) in the olfactory bulb (OB) 4 days post-OBI (olfactory bulb impact). Representative images from sham, contralateral, and ipsilateral OB are shown from left to right. Glial fibrillary acidic protein (GFAP) (A), cleaved caspase-3 (B), beta amyloid precursor protein (β-APP) (C), aquaporin 4 (AQP4) (D), and p75^NTR (E) showing a variable increases in TBI markers between ipsi- and contralateral OB with some changes extending into deeper layers. GCL, granule cell layer; EPL, external plexiform layer; GL, glomerular layer. Color image is available online at www.liebertpub.com/neu

FIG. 6.

Olfactory functional loss 4 days post-OBI (olfactory bulb impact). Olfactory behavior assays indicate a clear loss of function after traumatic brain injury. (A) The habituation/dishabituation assay utilizes an animal's innate ability to investigate novel odorants and is a measure of olfactory sensitivity and function. Mice are exposed to four trials in which they are presented with a small piece of filter paper. In the first three trials, mice are presented with water, and as the trials proceed, mice become gradually less interested in exploring the water-laced filter paper. This increasingly shorter investigation time to a sequentially presented stimulus is called habituation. Odor (amyl acetate) is presented in the fourth trial, and if the mice can detect it, they will resume investigating and sniffing the odor-laced paper (dishabituation). Sham mice clearly dishabituated and were able to smell the odor (compare trials 3 and 4; p<0.01). In contrast, the injured animals did not dishabituate, indicating that they are much less sensitive to the odor and possibly even anosmic (compare sham and OBI; p<0.01). (B) The buried food assay utilizes an animal's innate ability to forage and is a measure of olfactory sensitivity and function. After overnight food deprivation, mice were given 10 min to find a Pepperidge Farm fish buried under bedding. Sham littermates all found the food in less than the allotted time, unlike the injured animals that all failed. Importantly, these animals show no motor or cognitive impairments, as determined by their neurological severity scores (data not shown). This indicates that OBI mice have a lower olfactory sensitivity to the odor in the food presented because of their injury.

FIG. 7.

Markers of traumatic brain injury (TBI) in the olfactory epithelium (OE) and olfactory bulb (OB) after a 30-day recovery period. Representative images from sham, contralateral, and ipsilateral OB are shown from left to right. OE (A) and OB (B). Markers: p75^NTR; beta amyloid precursor protein (β-APP); cleaved caspase-3; growth-associated protein 43 (GAP-43); glial fibrillary acidic protein (GFAP); and aquaporin 4 (AQP4), revealing a reduction in some TBI markers while others persist. Color image is available online at www.liebertpub.com/neu

FIG. 8.

Partial return of olfactory circuitry and function after a 30-day recovery period. Histogram comparing olfactory sensory neuron (OSN) numbers of sham, contralateral side, and ipsilateral side (contra/Ipsi: p<0.02; compare with Fig. 2C). (A) Histogram of olfactory bulb (OB) and glomerular layer (GL) of partially recovered animals (sham/Ipsi: p<0.001; sham/contra: p<0.002 for OB; sham/ipsi: p<0.03; sham/contra: p<0.0001 for GL. Compare with Fig. 4D). (B) Sham mice dishabituated and were able to smell the odor (compare trials 3 and 4; p<0.04). (C) Similarly, the injured/recovered animals also dishabituated and were able to smell the odor (p<0.01; compare with Fig. 6A). (D) Sham and injured animals all found the food in less than the allotted time in the buried food assay. The injured animals improved, but were slower to find the buried food compared to sham animals (p<0.03; compare with Fig. 6B).

FIG. 9.

Effect of odor-induced activity on olfactory system recovery after TBI. Images show X-Gal staining of M71⁺ olfactory sensory neurons (OSNs) in turbinates 1 and 2 of M71-IRES-tauLacZ mice after 30 days of 10% acetophenone conditioning after injury. (A) Quantification of M71 OSNs in turbinates 1 and 2. Olfactory bulb impact (OBI)/no odor animals had greatest OSN number (vs. sham/aceto: p<0.0001; vs. control/no odor p<0.004). (B) X-Gal staining of M71 glomerulus on uninjured OB. (C) Glomerular volume of mice after 30 days of odor conditioning and controls. (D) Graph shows that OBI/no odor animals had largest glomerular volume (vs. sham/aceto: p<0.02). Color image is available online at www.liebertpub.com/neu