Willingness to vaccinate children against COVID-19 declined during the pandemic.

OBJECTIVES: To document the level of vaccine hesitancy in caregivers' of children younger than 12 years of age over the course of the pandemic in Pediatric Emergency Departments (ED). Study design Ongoing multicenter, cross-sectional survey of caregivers presenting to 19 pediatric EDs in the USA, Canada, Israel, and Switzerland during first months of the pandemic (phase1), when vaccines were approved for adults (phase2) and most recently when vaccines were approved for children (phase3). RESULTS: Willingness to vaccinate rate declined over the study period (59.7%, 56.1% and 52.1% in the three phases). Caregivers who are fully vaccinated, who have higher education, and those worried their child had COVID-19 upon arrival to the ED, were more likely to plan to vaccinate in all three phases. Mothers were less likely to vaccinate early in the pandemic, but this hesitancy attenuated in later phases. Older caregivers were more willing to vaccinate, and caregivers of older children were less likely to vaccinate their children in phase 3. During the last phase, willingness to vaccinate was lowest in those who had a primary care provider but did not rely on their advice for medical decisions (34%). Those with no primary care provider and those who do and rely on their medical advice, had similar rates of willingness to vaccinate (55.1% and 52.1%, respectively). CONCLUSIONS: COVID-19 vaccine hesitancy is widespread and growing over time, and public health measures should further try to leverage identified factors associated with hesitancy in order to enhance vaccination rates among children.

Serotonin projection patterns to the cochlear nucleus.

The cochlear nucleus is well known as an obligatory relay center for primary auditory nerve fibers. Perhaps not so well known is the neural input to the cochlear nucleus from cells containing serotonin that reside near the midline in the midbrain raphe region. Although the specific locations of the main, if not sole, sources of serotonin within the dorsal cochlear nucleus subdivision are known to be the dorsal and median raphe nuclei, sources of serotonin located within other cochlear nucleus subdivisions are not currently known. Anterograde tract tracing was used to label fibers originating from the dorsal and median raphe nuclei while fluorescence immunohistochemistry was used to simultaneously label specific serotonin fibers in cat. Biotinylated dextran amine was injected into the dorsal and median raphe nuclei and was visualized with Texas Red, while serotonin was visualized with fluorescein. Thus, double-labeled fibers were unequivocally identified as serotoninergic and originating from one of the labeled neurons within the dorsal and median raphe nuclei. Double-labeled fiber segments, typically of fine caliber with oval varicosities, were observed in many areas of the cochlear nucleus. They were found in the molecular layer of the dorsal cochlear nucleus, in the small cell cap region, and in the granule cell and external regions of the cochlear nuclei, bilaterally, of all cats. However, the density of these double-labeled fiber segments varied considerably depending upon the exact region in which they were found. Fiber segments were most dense in the dorsal cochlear nucleus (especially in the molecular layer) and the large spherical cell area of the anteroventral cochlear nucleus; they were moderately dense in the small cell cap region; and fiber segments were least dense in the octopus and multipolar cell regions of the posteroventral cochlear nucleus. Because of the presence of labeled fiber segments in subdivisions of the cochlear nucleus other than the dorsal cochlear nucleus, we concluded that the serotoninergic projection pattern to the cochlear nucleus is divergent and non-specific. Double-labeled fiber segments were also present, but sparse, in the superior olive, localized mainly in periolivary regions; this indicated that the divergence of dorsal and median raphe neurons that extends throughout regions of the cochlear nucleus also extended well beyond the cochlear nucleus to include at least the superior olivary complex as well.

Neurodegenerative changes in the guinea pig brainstem after intratympanic injection of gentamicin.

Gentamicin (4%) was injected intratympanically into the middle ear of guinea pigs and the effects on the brainstem evaluated 28 days later by immunohistochemistry with an antibody against glial fibrillary acidic protein to detect astrocytes, and by cytochrome oxidase staining to detect metabolic activity. Astrocytes were observed in the cochlear nucleus indicating the possible presence of anterograde degeneration. Deficiencies in the intensity of cytochrome oxidase staining up to the level of the superior olivary complex indicated the presence of abnormal metabolic activity. Both of these observations support the conclusion that a single intratympanic injection of gentamicin may lead to neuronal degeneration along the central auditory pathway.

Serotoninergic innervation of stapedial and tensor tympani motoneurons.

Retrograde tracing and neurotransmitter immunohistochemistry were combined to determine whether serotonin neurons innervated stapedial and tensor tympani motoneurons. With high-power light microscopy, putative axo-somatic and axo-dendritic contacts were observed between serotonin-positive endings and both stapedial and tensor tympani motoneurons, indicating that serotonin neurons terminate on brainstem motoneurons innervating the middle-ear muscles. With this connection, the serotonin system may directly modulate middle-ear muscle activity.

Time course of eye nystagmus and body movement after peripheral vestibular lesions in guinea pigs.

Body movement of guinea pigs was measured using a force platform at various times before and after unilateral end organ ablation and before and after sham surgery. Both spontaneous and drop-evoked movement patterns differed in the same animal after vestibular ablation and from control animals that received sham lesions. Whereas measures of eye nystagmus disappeared by 48 h postablation, measures of body movement indicated persistent differences even at 72 h. We conclude that the force platform can differentiate between movement patterns of normal and vestibular-lesioned animals and, in fact, measures a vestibular deficiency that is independent of eye nystagmus. The force platform appears to be a useful addition to evaluate vestibular deficits as well as to detect any benefits of pharmacological or surgical therapies.

Effect of pulsed electromagnetic stimulation on facial nerve regeneration.

OBJECTIVE: To determine if exposure to electromagnetic fields influences regeneration of the transected facial nerve in the rat. DESIGN AND METHODS: The left facial nerve was transected in the tympanic section of the fallopian canal in 24 rats randomly assigned to 2 groups. The cut ends of the facial nerve were reapproximated without sutures within the fallopian canal to maximize the potential for regeneration. Rats in the experimental group (n= 12) were then exposed to pulsed electromagnetic stimulation (0.4 millitesla at 120 Hz) for 4 hours per day, 5 days per week, for 8 weeks. Rats in the control group (n=12) were handled in an identical manner without pulsed electromagnetic stimulation. Four other rats were given sham operations in which all surgical procedures were carried out except for the actual nerve transection. Two of these rats were placed in each group. Nerve regeneration was evaluated using electroneurography (compound action potentials), force of whisker and eyelid movements, and voluntary facial movements before and at 2-week intervals after transection. Histological evaluation was performed at 10 weeks after transection. Each dependent variable was analyzed using a 2-way analysis of variance with 1 between variable (groups) and 1 within repeated measures variable (days after transection). RESULTS: Statistical analysis indicated that N1 (the negative deflection of depolarization phase of the muscle and/or nerve fibers) area, N1 amplitude, and N1 duration, as well as absolute amplitude of the compound action potentials, were all significantly greater 2 weeks after transection in the experimental than in the control group of rats. The force of eye and whisker movements after electrical stimulation was statistically greater in the experimental group of rats 4 weeks after transection. Voluntary eye movements in the experimental group were significantly better at 5 and 10 weeks, while whisker movements were better at 3 and 10 weeks. There was no statistical difference between the 2 groups for any histological variable. CONCLUSION: Results of this study indicate that pulsed electromagnetic stimulation enhances early regeneration of the transected facial nerve in rats.

Effects of para-chlorophenylalanine (pCPA) on the bush baby auditory brainstem response.

Click-evoked auditory brainstem responses were recorded in a prosimian primate, the bush baby (Otolemur garnettii), before and after depletion of serotonin (by systemic injection of para-chlorophenylalanine; pCPA) and up to 20 days after discontinuing pCPA injections (during the recovery of serotonin). Biphasic 100 micros clicks were presented at five repetition rates (13.2, 33.2, 53.2, 73.2, and 93.2 clicks/s; RATE) and sound pressure levels (SPL) were varied in 10 dB steps from 120-60 dB SPL peak equivalent. Absolute latencies of vertex-positive peaks I, III, IV, and V were measured from click onset. The latencies from each wave were statistically analyzed with a two-way analysis of variance using either RATE or SPL (but not both) and TIME AFTER pCPA as independent variables. Prior to pCPA, brainstem response latencies increased as a function of both decreasing SPL and increasing RATE. After pCPA, these normal increases in wave latency increased even more, particularly in response to high click rates. After pCPA was discontinued, measurements taken at weekly intervals indicated that latencies decreased after 1 week, increased to the highest values recorded after 2 weeks, and returned to normal after 3 weeks. These dynamic changes were interpreted to be the result of postsynaptic receptor up-regulation during the 10 days of continuous pCPA administration. These results suggest that serotonin plays an important role in sensory processing at the cellular level and, tonically, facilitates the auditory brainstem response to sound.

Light microscopic evidence of serotoninergic projections to olivocochlear neurons in the bush baby (Otolemur garnettii).

Double-label techniques were used to concomitantly label olivocochlear neurons and serotoninergic fibers in the bush baby (Otolemur garnettii) brainstem. Light-microscopic examination (using a 100 x plan apochromatic oil-immersion objective) of the sections revealed that serotonin-positive varicosities (presumptive terminal endings) contacted somata and dendrites of neurons belonging to both the lateral and medial olivocochlear neurons near the superior olivary nuclei. These results provide direct evidence that the olivocochlear system (a specific auditory brainstem pathway) receives input from the serotoninergic system (a diffuse reticular brainstem network).

Changes in brainstem calcitonin gene-related peptide after VIIth and VIIIth cranial nerve lesions in guinea pig.

The present study investigated the effect of seventh and eight cranial nerve lesions on the prominence of calcitonin gene-related peptide in the hypoglossal (XII), facial (VII), abducens (VI), and oculomotor (III) cranial nerve nuclei. Guinea pigs were anesthetized and subjected to unilateral cochlear removal, vestibular end organ ablation, and seventh nerve transection. After a survival period ranging from 4 h to 5 days, each animal was anesthetized and perfused intracardially. Frozen sections were collected through the brainstem and stained immunohistochemically for calcitonin gene-related peptide using a polyclonal antibody with the Vectastain ABC kit and protocol. Positive cells were counted in each nucleus bilaterally and analyzed for side to side differences. Nuclei XII and III showed no significant difference in the numbers of cells staining positively for calcitonin gene-related peptide between the ipsilateral and the contralateral sides to the lesion. However, nuclei VII and VI showed elevated numbers ipsilateral to the lesion on some days, but not all. For VII, there was no significant difference before 24 h, but there were significant differences 1-5 days after the lesion. Similarly, in VI, there was no difference before 24 h, but differences were significant beginning with day 1 and continuing through day 3, and finally disappearing by day 4. Changes in the numbers of CGRP positive cells in VII measurable 24 h after the lesion and continuing for at least 5 days afterward indicate a central nervous system retrograde response to peripheral motor nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and origin of serotoninergic afferents to guinea pig cochlear nucleus.

The distribution of serotoninergic fibers in the guinea pig cochlear nucleus was studied with serotonin immunohistochemistry. In addition, the origin of the serotoninergic fibers was determined by combining the retrograde transport of wheat germ agglutinin-apohorseradish peroxidase (gold conjugated) with serotonin immunohistochemistry. Immunoreactivity was present in varicose and nonvaricose fibers that were unevenly distributed throughout the cochlear nucleus. The fibers were most prominent in the superficial layers of the dorsal cochlear nucleus and the anterior spherical cell area of the anteroventral cochlear nucleus. Although less prominent, serotonin-positive fibers were also present in the remaining part of the anteroventral cochlear nucleus and the posteroventral cochlear nucleus. A few positive fibers were present in the auditory nerve root and the dorsal and intermediate acoustic striae. Double-labeled cells were found throughout the rostral-caudal extent of the serotoninergic system from the caudal linear nucleus to the nucleus raphe pallidus. However, most were confined to the dorsal (52%) and median (18%) raphe nuclei. Some serotoninergic cell groups contained retrogradely labeled cells that were not serotonin immunoreactive, indicating nonauditory afferents to cochlear nucleus containing other neurotransmitter substances. Serotonin may tonically modulate auditory processing within the cochlear nucleus as well as influence certain ascending auditory pathways. Most of the serotonin in the cochlear nucleus comes from superior raphe nuclei that also project to basal ganglia motor systems and limbic structures. Therefore, the effect of serotonin on the cochlear nucleus may be related to level of arousal or behavioral state.

Accumulation of the p53 tumor-suppressor gene product in oral leukoplakia.

OBJECTIVES: (1) To determine whether the protein of the suppressor gene p53 accumulates in leukoplakia of the oral cavity in individuals who use snuff; and (2) to determine whether a correlation exists between the accumulation of p53 protein and the degree of epithelial dysplasia present in oral leukoplakia. DESIGN: Retrospective analysis of archival tissue specimens. SETTING: The University Hospital, a tertiary referral hospital affiliated with the Oklahoma University Medical Center, Oklahoma City, Oklahoma. PATIENTS: In the first part of the study, biopsy specimens of leukoplakia from 12 persons who used snuff were compared with specimens from uninvolved oral mucosa of the same persons and with biopsy specimens from 12 nontobacco-using persons. In the second part of the study, accumulation of p53 protein was determined in 42 archival paraffin-embedded specimens from oral leukoplakia and correlated with the degree of epithelial dysplasia. METHODS: Accumulation of p53 protein was assessed by immunoperoxidase staining with four different primary antibodies. Positive cells were counted in five consecutive high-power fields. RESULTS: In part one, the average number of positive cells in the leukoplakia of snuff-users (21.89 +/- 4.33; mean +/- SE) was higher than that of normal-appearing mucosa (4.00 +/- 1.0; p < 0.05) and that of nontobacco-using controls (7.00 +/- 5.04). In part two, the average number of positive cells was higher in the moderately dysplastic (140.36 +/- 30.03) and severely dysplastic lesions (232.86 +/- 26.85) than in the mildly dysplastic lesions (14.53 +/- 3.33; p < 0.05). The correlation between the degree of epithelial dysplasia and the number of cells positive is strong (Spearman's correlation coefficient = 0.853). CONCLUSIONS: The accumulation of p53 protein in leukoplakia of snuff-users is higher than in normal-appearing oral mucosa from both snuff-users and nontobacco-using controls. A strong correlation exists between the degree of epithelial dysplasia present in oral leukoplakia and the number of cells staining positive for p53. The accumulation of p53 protein holds potential as an intermediate end point in studies of chemoprevention of oral cancer.

Serotonin and serotonin receptors in the central auditory system.

Immunohistochemical and ligand-binding techniques were used to visualize the neurotransmitter serotonin and one of its receptors, the 5-HT1A subtype, in auditory nuclei of the brainstem. Serotonergic fibers and terminal endings were found in all auditory nuclei extending from the cochlear nucleus to the inferior colliculus, including the superior olivary complex and the nuclei of the lateral lemniscus. The density of the innervation varied between and within each nucleus. All serotonergic cell bodies were located outside the auditory nuclei. The 5-HT1A receptor subtype was found in the cochlear nucleus as well as in the inferior colliculus. With no serotonergic cell bodies present in the auditory nuclei, the present neuroanatomic and neurochemical findings support behavioral and neurophysiologic findings that the serotonergic system may modulate central auditory processing.

Relationship of descending inferior colliculus projections to olivocochlear neurons.

With the objective of defining the relationship of descending inferior colliculus projections to the olivocochlear system in the guinea pig, inferior colliculus neurons were anterogradely labeled with Phaseolus vulgaris-leucoagglutinin and olivocochlear neurons were retrogradely labeled with horseradish peroxidase in the same brain sections. Inferior colliculus neurons were found to project to many nuclei and regions of the hindbrain where olivocochlear neurons reside. The most substantial of these descending projections was to the ipsilateral medioventral periolivary region. Fewer descending projections terminated in the ipsilateral ventral nucleus of the lateral lemniscus, superior paraolivary nucleus, and rostral periolivary region; and even fewer ipsilateral projections terminated in the area surrounding the lateral superior olive, caudal periolivary region, and the lateroventral periolivary region. Descending neurons of the inferior colliculus also project to the contralateral hindbrain first via the lateral lemniscus and then the trapezoid body, to terminate in the contralateral medioventral periolivary region, superior paraolivary nucleus, rostral periolivary region, and the ventral nucleus of the lateral lemniscus. In addition to the projections into these regions that contain olivocochlear neurons, there are varicosities of inferior colliculus neurons that appear to contact the olivocochlear neurons themselves, both ipsilaterally and contralaterally, especially, but not only, in the ipsilateral medioventral periolivary region. We therefore conclude that descending inferior colliculus neurons do provide input to olivocochlear neurons and that the input is not limited to olivocochlear neurons of the ipsilateral medioventral periolivary region. However, given the robust nature of the projection to the ipsilateral medioventral periolivary region and the paucity of contacts observed in that region, we also conclude that the olivocochlear neuron is not the major target of descending inferior colliculus projections.

Markers of keratinocyte differentiation in snuff-induced leukoplakia.

Biopsy specimens from 12 patients who used snuff and had leukoplakia in the mucosa of the oral cavity were studied and compared with specimens from their own nonleukoplakic oral mucosa, as well as with biopsy specimens from corresponding areas of the oral cavity in 12 nonsmoking, nontobacco-using control subjects. The biopsy specimens were processed using standard immunohistochemical rabbit antibody to human involucrin and mouse antibody to human transglutaminase type I as the primary antibodies. A computer-driven light absorbance image analysis system was used to determine the optical density of each of the marker-stained specimens. Optical density measurements were compared using a one-way analysis of variance. The expression of involucrin was significantly higher in the epithelium of the nonsmoking, nontobacco-using control subjects (0.2937 +/- 0.0725 optical density) in comparison with the normal-appearing mucosa (0.2283 +/- 0.0488 optical density) and the leukoplakic mucosa of the snuff-using patients (0.2007 +/- 0.0669 optical density) (p < 0.05). The expression of transglutaminase type I was also significantly higher in the epithelium of the nonsmoking, nontobacco-using controls (0.2308 +/- 0.1381 optical density) than in the patients with leukoplakic mucosa (0.1310 +/- 0.0472 optical density) (p < 0.05). However, there was no difference when compared with the normal-appearing mucosa of the patients in the snuff-using group (0.1686 +/- 0.0323 optical density). This study has shown that involucrin and transglutaminase type I are expressed differently in leukoplakic oral mucosa of snuff users and in normal oral mucosa and that this difference can be measured objectively.

Projections from the posteroventral cochlear nucleus to the superior olivary complex in guinea pig: light and EM observations with the PHA-L method.

The efferent neural projections from posteroventral cochlear nucleus to the superior olivary complex in guinea pig were examined with the Phaseolus vulgaris-leucoagglutinin anterograde tract-tracing method. Light microscopic analysis demonstrated that the posteroventral cochlear nucleus of guinea pig bilaterally projects to the superior para-olivary nucleus and the rostral, medioventral, and lateroventral peri-olivary regions. Ipsilaterally, the posteroventral cochlear nucleus projects to the lateral superior olive, the caudal peri-olivary region, and areas immediately surrounding the capsule of the lateral superior olive. Contralaterally, the posteroventral cochlear nucleus projects to the medial nucleus of the trapezoid body. All of these projection axons travel in the trapezoid body and their terminals make, primarily, en passant endings upon their targets. Exclusively within the contralateral medial nucleus of the trapezoid body, some neurons terminate also in calyceal endings. The assumption that immunolabeled structures observed with light microscopy made actual synaptic contact in their respective target areas was confirmed with electron microscopy. With postembedding immunocytochemical procedures applied to thin sections, the electron microscope revealed labeled synaptic vesicles and pre- and postsynaptic membrane specializations. The projection pattern from posteroventral cochlear nucleus was found to be topographically organized in three distinct regions. The more dorsally located neurons of the posteroventral cochlear nucleus terminated dorsomedially in the ipsilateral lateral superior olive, ventromedially in the contralateral superior para-olivary nucleus, and medially in the contralateral medioventral peri-olivary region. The more ventrally located neurons of the posteroventral cochlear nucleus terminated dorsolaterally in the ipsilateral lateral superior olive, dorsolaterally in the contralateral superior para-olivary nucleus, and laterally in the contralateral medioventral peri-olivary region.

Posteroventral cochlear nucleus projections to olivocochlear neurons.

The presence of ascending auditory inputs from the posteroventral cochlear nucleus (PVCN) to olivocochlear neurons was examined in guinea pig by using the combination Phaseolus vulgaris-leucoagglutinin (PHA-L) anterograde and horseradish peroxidase (HRP) retrograde tract-tracing technique. By labeling the somata of olivocochlear neurons after injection of HRP into the cochlea and simultaneously labeling terminal endings of PVCN efferent neurons after injection of PHA-L into PVCN, we observed neuronal connections between these two elements within all regions of the superior olivary complex known to contain olivocochlear neurons. These regions include the superior paraolivary nucleus, medial nucleus of the trapezoid body, lateral superior olive, and periolivary regions. All possible projection patterns regarding side of input and output of both large (four combinations) and small (two combinations) olivocochlear neurons were observed. However, the most frequently observed pattern was the PVCN projection to a contralaterally located and contralaterally projecting, large olivocochlear neuron. Thus the most prevalent pattern demonstrated a feedback pathway that crossed the brainstem twice. Additional patterns demonstrated pathways that fed back to the same cochlea as well as pathways that fed forward to the opposite cochlea.

Immunocytochemical study of the GABA system in chicken vestibular endorgans and the vestibular ganglion.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA), GABA synthesizing enzyme; L-glutamate decarboxylase (GAD) and degradative enzyme; GABA transaminase (GABA-T) in the chicken vestibular endorgans and the vestibular ganglion was investigated. GABA and GAD-like immunoreactivity were confined to the sensory hair cell cytoplasm, suggesting that GAD synthesizes GABA in the hair cell. GABA-T-like immunoreactivity, indicative of GABA degradation, was found around hair cells, along nerve fibers running through the stroma and within the ganglion cell. These immunocytochemical findings indicate that the GABAergic system exists in the chicken vestibular endorgans and that GABA may function as an afferent neurotransmitter at the level of hair cells.

Neural connections identified with PHA-L anterograde and HRP retrograde tract-tracing techniques.

This paper describes a method of identifying specific input and output elements of a two-neuron projection pathway. Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde tract-tracing was used in combination with the retrograde transport of horseradish peroxidase (HRP) to demonstrate connections between small groups of neurons in the brainstem auditory system. Specifically, the projection from cochlear nucleus to olivary neurons that project to the cochlea were demonstrated. The first neuron in this pathway (the cochlear nucleus neuron) was anterogradely labelled with PHA-L and could be traced via labelled axons and terminals to the second neuron (the olivocochlear neuron) whose soma was labelled with HRP after cochlear injection.

Light- and electron-microscopic study of gamma-aminobutyric-acid-like immunoreactivity in the guinea pig organ of Corti.

With light and electron microscopy, gamma-aminobutyric-acid (GABA)-like immunoreactivity was examined in the guinea pig organ of Corti. In whole-mount preparations, although GABA-like immunoreactivity was present within efferent components in all turns of the cochlea, it was more intense apically. At the ultrastructural level, GABA-like immunoreactivity was clearly restricted to the efferent system, appearing in axons of the inner and tunnel spiral bundles, in axons crossing the tunnel of Corti, and in terminals filled with numerous labeled vesicles synapsing on outer hair cells.

Immunocytochemical study of catecholaminergic innervation in the guinea pig cochlea.

The enzymes for synthesis of catecholamine, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyl transferase (PNMT), have been immunocytochemically localized in the guinea pig cochlea. Two groups of catecholamine-containing neurons could be distinguished. The first group, which was TH-positive/DBH, PNMT-negative, was found in the inner spiral bundle and the tunnel spiral bundle. The other group was TH, DBH-positive/PNMT-negative, and was found around the blood vessels. The immunocytochemical evidence obtained in this experiment suggests that the catecholamines should play a functional role in the guinea pig cochlea.