Alan M. Robinson

Pathology of the Olfactory Mucosa: Implications for the Treatment of Olfactory Dysfunction

Objective: The pathology of the olfactory mucosa is poorly understood; however, most cases of hyposmia and anosmia appear to be associated with a decline in the number of functioning mature olfactory sensory neurons (OSNs). Under normal conditions, OSNs undergo apoptotic cell death at a baseline rate likely secondary to their exposed location in the nose. Regeneration of mature OSNs from precursors in the epithelium allows the animal to maintain an adequate number of neurons necessary for olfactory sensation. In many cases of olfactory dysfunction, this balance is apparently disturbed, with a net loss of OSNs. The current study will examine normal and diseased olfactory tissue for the presence of data demonstrating that the preferred mechanism of OSN cell death is apoptotic in both health and disease. The potential therapeutic implications will be discussed.

Optical cochlear implants: evaluation of surgical approach and laser parameters in cats.

Previous research has shown that neural stimulation with infrared radiation (IR) is spatially selective and illustrated the potential of IR in stimulating auditory neurons. The present work demonstrates the application of a miniaturized pulsed IR stimulator for chronic implantation in cats, quantifies its efficacy, and short-term safety in stimulating auditory neurons. IR stimulation of the neurons was achieved using an optical fiber inserted through a cochleostomy drilled in the basal turn of the cat cochlea and was characterized by measuring compound action potentials (CAPs). Neurons were stimulated with IR at various pulse durations, radiant exposures, and pulse repetition rates. Pulse durations as short as 50 mus were successful in evoking CAPs in normal as well as deafened cochleae. Continual stimulation was provided at 200 pulses per second, at 200 mW per pulse, and 100 mus pulse duration. Stable CAP amplitudes were observed for up to 10 h of continual IR stimulation. Combined with histological data, the results suggest that pulsed IR stimulation does not lead to detectable acute tissue damage and validate the stimulation parameters that can be used in future chronic implants based on pulsed IR.

Pathology of the olfactory epithelium: Smoking and ethanol exposure

Objective: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis.

Spread of cochlear excitation during stimulation with pulsed infrared radiation: Inferior colliculus measurements

Infrared neural stimulation (INS) has received considerable attention over the last few years. It provides an alternative method to artificially stimulate neurons without electrical current or the introduction of exogenous chromophores. One of the primary benefits of INS could be the improved spatial selectivity when compared with electrical stimulation. In the present study, we have evaluated the spatial selectivity of INS in the acutely damaged cochlea of guinea pigs and compared it to stimulation with acoustic tone pips in normal-hearing animals. The radiation was delivered via a 200 µm diameter optical fiber, which was inserted through a cochleostomy into the scala tympani of the basal cochlear turn. The stimulated section along the cochlear spiral ganglion was estimated from the neural responses recorded from the central nucleus of the inferior colliculus (ICC). ICC responses were recorded in response to cochlear INS using a multichannel penetrating electrode array. Spatial tuning curves (STCs) were constructed from the responses. For INS, approximately 55% of the activation profiles showed a single maximum, ∼22% had two maxima and ∼13% had multiple maxima. The remaining 10% of the profiles occurred at the limits of the electrode array and could not be classified. The majority of ICC STCs indicated that the spread of activation evoked by optical stimuli is comparable to that produced by acoustic tone pips.

Apoptosis in the Aging Olfactory Epithelium

Objectives Olfactory receptor neurons undergo apoptosis at a baseline rate, probably secondary to environmental damage even in the absence of gross disease. The study demonstrates age‐related changes in expression of genes known to regulate apoptosis in the rat olfactory mucosa. These results are compared with gene expression in young rats and rats that have undergone surgical deafferentation of the olfactory receptor neurons.

Expression of glucocorticoid receptor mRNA and protein in the olfactory mucosa: Physiologic and pathophysiologic implications

Objectives: Define the presence and distribution of glucocorticoid receptors (GRs) within the olfactory mucosa in order to assess potential physiologic and pathophysiologic effects of these hormones on olfaction. Study Design: The olfactory mucosa was harvested from adult male rats and guinea pigs. Kidney tissue was utilized as a known positive control. Methods: The techniques of reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunocyto‐chemistry were utilized to examine the expression of GR mRNA and protein. To assure the presence of olfactory mucosa in the nasal tissue samples, RT‐PCR was utilized to identify the olfactory marker protein (OMP). Results: The presence of GR mRNA was confirmed in both the olfactory mucosa and kidney. GR‐like immunoreactivity associated with the olfactory epithelium was greatest at the apical surface, a position corresponding to the dendrites, knobs, and cilia of olfactory receptor neurons, as well as the supranuclear region of sustentacular cells. Weaker GR‐like immunoreactivity was associated with the region of the cell bodies of the olfactory receptor neurons. Within the lamina propria, acinar cells of the Bowmans glands and olfactory nerve bundles were intensely immunoreactive. Conclusions: The presence of GR mRNA and protein within the olfactory mucosa is consistent with a functional role for glucocorticoid hormones in the systemic regulation of olfaction. Furthermore, these studies suggest that corticosteroid medications may have direct effects on the cells of the olfactory mucosa in the pathologic state. The potential mechanisms whereby these hormones may act are discussed.

Age-related olfactory dysfunction: cellular and molecular characterization in the rat.

Background Olfactory receptor neurons (ORNs) undergo apoptosis at a baseline rate even in the absence of obvious disease. Although the precise triggers of the apoptotic cascade are unclear, ORNs are exposed directly to the external environment, making them susceptible to injury. As an adaptive mechanism, mammals have the ability to replace lost ORNs throughout adult life from neuronal precursors within the olfactory epithelium (OE). In humans, this process fails with age as the surface area of the OE and the number of ORNs decline, coupled with a loss of clinical olfactory function. The question addressed in this study is whether this age-related failure of olfactory sensation is a result of a decrease in neuronal proliferation or an increase in ORN cell death. Methods To begin to address this question the ribonuclease protection assay was used to assess expression of apoptosisrelated genes in rat OE as a function of age. Second, the terminal deoxynucleotide transferase end labeling assay was used to assess the percentage of ORNs undergoing apoptosis (apoptotic index) in three groups of animals: young (12 weeks), old (32 months), and bulbectomized rats. Bulbectomy is a standard model for ORN injury associated with a massive increase in ORN apoptosis and serves as a positive control. Results Ribonuclease protection assay data indicate an age-related increase in Bax, Bcl-xL, and procaspase-3 messenger RNA expression in aged compared with young rats. A similar but more pronounced increase in expression of these apoptotic-related genes is seen after bulbectomy. The terminal deoxynucleotide transferase end labeling assay also showed a statistically significant increase in the apoptotic index with both age and bulbectomy. Conclusion Taken together, the current results indicate that aging and injury induce parallel changes in OE. Furthermore, these findings support the hypothesis that age-related olfactory dysfunction is, at least in part, related to an increase in ORN cell death.

Infrared neural stimulation: Beam path in the guinea pig cochlea

It has been demonstrated that INS can be utilized to stimulate spiral ganglion cells in the cochlea. Although neural stimulation can be achieved without direct contact of the radiation source and the tissue, the presence of fluids or bone between the target structure and the radiation source may lead to absorption or scattering of the radiation, which may limit the efficacy of INS. The present study demonstrates the neural structures in the radiation beam path that can be stimulated. Histological reconstructions and microCT of guinea pig cochleae stimulated with an infrared laser suggest that the orientation of the beam from the optical fiber determined the site of stimulation in the cochlea. Best frequencies of the INS-evoked neural responses obtained from the central nucleus of the inferior colliculus matched the histological sites in the spiral ganglion.

Behavioral and Electrophysiological Responses Evoked by Chronic Infrared Neural Stimulation of the Cochlea

Infrared neural stimulation (INS) has been proposed as a novel method for neural stimulation. In order for INS to translate to clinical use, which would involve the use of implanted devices over years or decades, the efficacy and safety of chronic INS needs to be determined. We examined a population of cats that were chronically implanted with an optical fiber to stimulate the cochlea with infrared radiation, the first known chronic application of INS. Through behavioral responses, the cats demonstrate that stimulation occurs and a perceptual event results. Long-term stimulation did not result in a change in the electrophysiological responses, either optically-evoked or acoustically-evoked. Spiral ganglion neuron counts and post implantation tissue growth, which was localized at the optical fiber, were similar in chronically stimulated and sham implanted cochleae. Results from chronic INS experiments in the cat cochlea support future work toward INS-based neuroprostheses for humans.

Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing.

BACKGROUND Tissue ischemia and aging are independent features associated with the healing impairment of cutaneous wounds. However, the pathophysiology of these processes as they relate to impaired-healing wounds is poorly understood. MATERIALS AND METHODS A single full-thickness biopsy wound was made on both ears of young (3-6 month) and aged (>24 month) Fisher rats. One ear was rendered ischemic by transection of the vasculature at the ear base, while the other ear served as an internal nonischemic control. Wounds were harvested from 3 to 7 days and were evaluated histologically for either granulation tissue formation and epithelialization. Total RNA from wounds harvested at postoperative day 7 was probed using a nylon-based cDNA array to assess global genetic expression alterations. RESULTS Healing in the rat ear model is impaired by both ischemia and advanced age as measured by granulation tissue formation and wound epithelialization. Granulation tissue formation was affected to a greater degree by ischemia than age (-58% versus -21%, respectively) while epithelialization displayed an opposite response (-17% versus -53%, respectively). Global analysis of gene expression suggests that ischemia engenders a marked increase in genes displaying altered expression in aged animals compared to young animals. Importantly, all possible alterations in gene expression are found in samples from aged ischemic wounds, indicating that gene regulation is not simply depressed by advanced age. CONCLUSIONS Wound epithelialization appears to be affected to a greater degree by advanced age than by ischemia. The results demonstrate the distinctive phenotype presented by the clinically relevant combination of age and ischemia in an in vivo model of cutaneous wound healing.