Questions about quitting (Q2): design and methods of a Multiphase Optimization Strategy (MOST) randomized screening experiment for an online, motivational smoking cessation intervention.

Effective interventions are needed to improve smokers' motivation for quitting, treatment utilization, and abstinence rates. The Internet provides an ideal modality for delivering such interventions, given the low cost, broad reach, and capacity to individually tailor content, but important methodological questions remain about how to best design and deliver an online, motivational intervention to smokers. The current paper reports on the intervention, study design and research methods of a randomized trial (called Questions about Quitting) designed to address some of these questions. Using a Multi-phase Optimization Strategy (MOST) screening experiment, the trial has two key aims: to examine the impact of four experimental intervention factors (each evaluated on two levels) on smokers' subsequent treatment utilization and abstinence, and to examine select moderators of each sub-factor's effectiveness. The experimental factors of interest are: navigation autonomy (content viewing order is dictated based on stage of change or not), use of self-efficacy based testimonials (yes vs. no), proactive outreach (reminder emails vs. no emails), and decisional framework (prescriptive vs. motivational tone). To our knowledge, this is the first application of the MOST methodology to explore these factors or to explore the optimal design for a motivational intervention targeting smokers not actively trying to quit smoking. The rationale for the experimental factor choice, intervention design, and trial methods are discussed. Outcome data are currently being collected and are not presented, but recruitment data confirm the feasibility of enrolling smokers at varying stages of readiness to quit.

Using microscopy to assess chorion structural integrity and parasitoid oviposition sites on stink bug (Hemiptera: Pentatomidae) eggs.

Previous efficacy studies found that many insecticides used by growers could be having an adverse effect on egg parasitoids (Telenomus podisi) developing in the eggs of the brown stink bug (Euschistus servus), while unhatched stink bugs experienced lower levels of mortality. One plausible explanation for this was that insecticides might enter parasitized eggs more readily via oviposition wounds. Parasitized E. servus eggs, as well as nonparasitized stink bug (Acrosternum hilare, E. servus, Murgantia histrionica, and Podisus maculiventris) eggs, were examined using electron microscopy. Egg response to perforation by a tungsten probe served as a control. Microscopy images depicted the chorion surface as characterized by a matrix of ridges and micropylar processes in a ring around the margin of the operculum. Observations of oviposition sites showed a "scab" formed where the ovipositor penetrated the chorion, and at sites penetrated by the probe. These formations appeared to be the result of fluids from inside the egg leaking out, drying, and hardening after oviposition or probe perforation, suggesting that the response was not due to substances secreted by the parasitoid. Further, no open wounds or holes were seen to increase the possibility of insecticides entering parasitized eggs.

Effects of columella removal on inner ear morphology in the chick.

We are currently removing the single middle ear bone (columella) in the domestic chick to introduce chemical agents directly into the inner ear. Since we are interested in the effect of these agents on neural structures within the avian basilar papilla (BP), we are concerned about any subtle changes that might result from the surgical procedure of columella removal alone. The purpose of this study was to use light and transmission electron microscopy to analyze morphological changes in the inner ear after columella removal. Fifteen-day-old chicks underwent a unilateral, bilateral or a sham removal of the columella. After columella removal, the oval window was either plugged with Gelfoam or Kimwipe (standard accepted procedure to prevent possible perilymph leak) or left uncovered. After a 5-day survival period, morphological changes were observed in the tegmentum vasculosum (TV) of all ears receiving a columella removal as compared to unoperated ears. Further, ears with Gelfoam plugging the oval window also had damage to the hair cells and support cells of the basilar papilla. In contrast, there were no observable differences in either auditory afferent or efferent nerve terminals on hair cells in the BP from any ears that had the columella removed compared to those from unoperated ears. These results suggest that columella removal alone may produce morphological changes to the TV within 5 days of surgery but not to structures within the BP. On the other hand, columella removal with a Gelfoam plug results in damage not only to the TV but also to cells within the basilar papilla during this same survival time. Despite damage to other structures within the inner ear, cochlear efferent and afferent terminals on surviving hair cells were unaffected by columella removal with or without plugging.

Recovery of noise-induced changes in the dark cells of the quail tegmentum vasculosum.

Morphologic changes in the tegmentum vasculosum (TV) of adult quail after high intensity sound exposure were studied. Quail were continuously exposed to 115 dB SPL, 1500 Hz pure tone in a sound field for 12 h and either sacrificed immediately (0 day), 1, 2, 3, 4, 6 or 10 days later. Serial sections through the basilar papilla at 100 micron intervals from base to apex were obtained for study with light microscopy and TEM. Significant morphologic changes were found within the TV of quail sacrificed on days 0-4. On a quantitative scale, the majority of recovery occurred within the first 24 h. After four days survival the tegmentum appeared nearly normal. This recovery correlates well with the temporal pattern of threshold shift recovery. These results demonstrate a temporal correlation between ultrastructural changes in the TV and functional recovery of hearing after intense sound exposure. A potential etiologic role of the TV in avian temporary threshold shift is suggested.

TEM analysis of neural terminals on autoradiographically identified regenerated hair cells.

Regenerated tall and short hair cells identified by autoradiography ([3H]thymidine) were analyzed for their neural contacts using transmission electron microscopy. Ears from mature Coturnix quail (N = 5) exposed to pure tone overstimulation (1500 Hz, 115 dB, 12 h) and treated with [3H] thymidine for 10 days were fixed, embedded, sectioned serially in 100 mu intervals and prepared for autoradiography. At fifty percent length along the papilla, alternating semi-thick (1 micron) and thin (70 nm) sections were taken at 50 microns intervals. Semi-thick sections were analyzed at the light microscope level for autoradiographic labeling of [3H]thymidine over the hair cell nucleus. When an autoradiographically labelled hair cell was identified the corresponding serial thin sections were analyzed in the transmission electron microscope. Seven autoradiographically labeled hair cells in semi-thin sections were positively identified in immediately adjacent thin serial sections. Labeled hair cells were morphologically similar to adjacent cells with no label and generally appeared to receive similar innervation. Regenerated short hair cells showed large chalice shaped, efferent terminals, intermediate hair cells received both afferent and efferent innervation and tall hair cells were contacted by two to three afferent terminals with synaptic specializations. These results provide conclusive evidence of both efferent and afferent synaptic contacts on newly regenerated hair cells of all types 10 days following acoustic trauma.

Changes in the acoustic nerve after hair cell regeneration.

Hair cells of the avian inner ear have been shown to regenerate following acoustic or ototoxic insult. The consequences of this regeneration on the acoustic nerve have yet to be defined. The purpose of the present study was to use TEM analysis following cochlear damage and hair cell regeneration to describe afferent and efferent neural terminals on hair cells in the newly repopulated sensory epithelium. Following acoustic overstimulation (12 h, 115 dB SPL, 1500 Hz) adult quail were sacrificed immediately (0 day), or at 2, 12, or 24 weeks. Serial thin sections were taken from the embedded papilla in a plane tangential to the basilar membrane in the area consistent with regenerative activity. Immediately following noise exposure very few hair cells could be seen within the epithelia; afferent terminals on remaining cells appeared normal. Two weeks later afferent terminals showed signs of degeneration; efferent terminals were rarely seen on tall hair cells but remained relatively normal on short hair cells. Three to six months later afferent terminals had regained a more normal appearance but were less numerous on tall hair cells; some return of efferent-like terminals was seen often contacting two tall hair cells. Large normal appearing, efferent terminals remained on short hair cells. These results suggest that regenerated hair cells are likely to receive neural innervation. It would appear that some degeneration of afferent terminals takes place prior to final innervation of new hair cells.

Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity.

Hair cell loss in the avian cochlea partially recovers following both acoustic trauma and aminoglycoside intoxication. DNA labeling with tritiated thymidine has shown that the restoration of cell number following acoustic trauma results from the production of new hair cells by mitotic division. The purpose of the present study was to determine if mitosis also contributes to the recovery of hair cell number which occurs following aminoglycoside intoxication. Chickens received daily injections of either gentamicin sulfate or distilled water for 10 consecutive days. During the latter 7 days of this period, all birds were also injected with [3H]thymidine. Following postinjection survival periods of 3 or 6 days, one papilla from each bird was processed for autoradiography and the other for scanning electron microscopy (SEM). Incorporation of [3H]thymidine was seen over hair cells and support cells in experimental papillae in regions of hair cell loss. No labeling was seen outside of damaged regions or in the papillae of control birds. SEM showed that damaged regions in experimental birds contained cells similar in appearance to developing auditory hair cells in avian embryos. These results show that the restoration of hair cell number following aminoglycoside toxicity results from the production of new cells by mitosis.

Hair cell regeneration in senescent quail.

Hair cell regeneration was studied following exposure to an intense pure tone stimulus in young adult and senescent Coturnix quail. Three, 3-month old and four, 3-year old quail were continuously exposed to a 1500 Hz pure tone at 115 dB SPL for 12 h. Four quail were not noise exposed and were used as age-matched controls. Control and experimental birds received injections of [3H]thymidine daily for 10 days after noise exposure. Ten days after noise exposure birds were killed and their cochleae embedded, sectioned serially and processed through standard methods of autoradiography. Hair cell counts showed a discreet area of hair cell loss for both age groups in the proximal half of the papilla. Incorporation of [3H]thymidine was clearly seen over the nuclei of hair cells and support cells in the region of hair cell loss in both age groups. Incorporation of [3H]thymidine was also seen over the nuclei of hair cells and support cells in a very small area in two of the non-exposed control birds. These results demonstrate that the potential for hair cell regeneration is maintained throughout life in Coturnix quail. Further, they suggest that there may be some very low level of hair cell production in the normal adult quail ear which is activated in the absence of massive trauma.

Ganglion cell loss continues during hair cell regeneration.

Hair cells and ganglion cells were counted in young adult quail (Coturnix coturnix) after acoustic trauma at 10, 30, 60 and 90 day survival times. Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 mu intervals from basal to apical tip. Hair cells and ganglion cells were counted from 3 mu thick sections at each interval. Hair cells were designated as tall or short within the area 30-70% of length from basal tip of the papilla. Both tall and short hair cells were significantly reduced in number 10 days following trauma. Tall hair cells recovered to within 96% of normal after 60-90 days. Short hair cells recovered but to a lesser extent. Ganglion cell loss did not begin until 30 days after trauma and continued without recovery 90 days after trauma. A good correlation was found for position of both types of hair cell loss and position of ganglion cell loss. These results suggest that the initial loss of hair cells, both tall and short, results in retrograde degeneration of neural fibers and ganglion cells.

Ganglion cell and hair cell loss in Coturnix quail associated with aging.

Hair cells and ganglion cells were examined in young adult (3 month old) and senescent (3 to 6 year old) quail (Coturnix coturnix). Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 micron intervals from basal to apical tip. Hair cells and ganglion cells were counted from three micron thick sections at each interval. Hair cell number remained constant between age groups (less than 10% loss even in the oldest group). Ganglion cell number, on the other hand, was considerably reduced in the senescent birds (20-60% loss). These results are similar to quantitative results in senescent mammals and suggest that ganglion cell loss may be generalized response to aging.

Topography of nonneoplastic and neoplastic cells of common origin.

The possibility that neoplastic transformation may characteristically alter cell surface morphology prompted a comparison by scanning electron microscopy of nonneoplastic and tumorigenic cell lines from a single clone of mouse embryo cells. Among those studied by scanning electron microscopy, six lines of this clone proved nonneoplastic, and nine others underwent neoplastic transformation in culture, as evidenced by tumor production in vivo. Combined cinephotomicrography and scanning electron microscopy allowed the determination of postmitotic time and topography of individual cells without perturbing the cells or detectably altering their surface morphology; no pattern of morphological change as a function of postmitotic time was evident in either nonneoplastic or neoplastic cell populations. Accordingly, these cell populations could be compared under their usual conditions of attached asynchronous growth despite differences in proliferation rates. Cells of the neoplastic lines were characteristically less spread, and some lines displayed greater morphological variability than was evident among cells of nonneoplastic lines. However, most cells in all nine neoplastic lines and all six nonneoplastic lines were smooth surfaced. Thus, the exaggerated incidence of microvilli, ruffles, or blebs reported for established tumor-derived lines and most morphologically transformed lines did not prove a reliable criterion of neoplastic state for these cell lines of common origin grown under the same culture conditions.