Role of cell cycle regulators in development of the inner ear

The inner ear originates from an ectodermal thickening called the otic placode. The otic placode invaginates and closes to an otic vesicle, the otocyst. The otocyst epithelium undergoes morphogenetic changes and cell differentiation, leading to the formation of the labyrinth-like mature inner ear. Epithelial-mesenchymal interactions control inner ear morphogenesis, but the modes and molecules are largely unresolved. The expressions of negative cell cycle regulators in the epithelium of the early-developing inner ear have also not been elucidated. The mature inner ear comprises the hearing (cochlea) and balance (vestibular) organs that contain the nonsensory and sensory cells. In mammals, the inner ear sensory cells, called hair cells, exit the cell cycle during embryogenesis and are mitotically quiescent during late-embryonic differentiation stages and postnatally. The mechanisms that maintain this hair cell quiescense are largely unresolved. In this work I examined 1) the epithelial-mesenchymal interactions involved in inner ear morphogenesis, 2) expression of negative cell cycle regulators in the epithelium of the early developing inner ear and 3) the molecular mechanisms that maintain the postmitotic state of inner ear sensory cells. We observed that during otocyst stages, epithelial fibroblast growth factor 9 (Fgf9) communicates with the surrounding mesenchyme, where its receptors are expressed. Fgf9 inactivation leads to reduced proliferation of the surrounding vestibular mesenchyme and to the absence of semicircular canals. Semicircular canal development is blocked, since fusion plates do not form. These results show that the mesenchyme directs fusion plate formation and give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of proliferation. We show that the members of the Cip/Kip family of CKIs (p21Cip1, p27Kip1 and p57Kip2) are expressed in the early-developing inner ear. Our expression data suggest that CKIs divide the otic epithelium into proliferative and nonproliferative compartments that may underlie shaping of the otocyst. At later stages, CKIs regulate proliferation of the vestibular appendages, and this may regulate their continual growth. In addition to restricting proliferation, CKIs may play a role in regional differentiation of various epithelial cells. Differentiating and adult inner ear hair cells are postmitotic and do not proliferate in response to serum or mitogenic growth factors. In our study, we show that this is the result of the activity of negative cell cycle regulators. Based on expression profiles, we first focused on the retinoblastoma (Rb) gene, which functions downstream of the CKIs. Analysis of the inner ear phenotype of Rb mutant mice show, that the retinoblastoma protein regulates the postmitotic state of hair cells. Rb inactivation leads to hyperplasia of vestibular and cochlear sensory epithelia that is a result of abnormal cell cycle entry of differentiated hair cells and of delayed cell cycle exit of the hair cell precursor cells. In addition, we show that p21Cip1 and p19Ink4d cooperate in maintaining the postmitotic state of postnatal auditory hair cells. Whereas inactivation of p19Ink4d alone leads to low-level S-phase entry (Chen et al., 2003) and p21Cip1 null mutant mice have a normal inner ear phenotype, codeletion of p19Ink4d and p21Cip1 triggers high-level S-phase entry of auditory hair cells during early postnatal life, which leads to supernumerary hair cells. The ectopic hair cells undergo apoptosis in all of the mutant mice studied, DNA damage being the immediate cause of this death. These findings demonstrate that the maintenance of the postmitotic state of hair cells is regulated by Rb and several CKIs, and that these cell cycle regulators are critical for the lifelong survival of hair cells. These data have implications for the future design of therapies to induce hair cell regrowth.

Modern surgical treatment of otosclerosis

The modern unilateral surgical treatment of otosclerosis started in 1956. Simultaneous bilateral surgery has not been reported in stapes surgery and in case of bilateral otosclerosis ears are operated in two different sessions. Simultaneous surgery would give the patient the opportunity to gain advantages of bilateral hearing within one session, with less time spent in hospital and on sick leave. The mechanism for vestibular symptoms and the exact end organ affected after surgery is still unveiled. This thesis presents the results of experimental simultaneous bilateral stapes surgery, and vestibular symptoms and findings before and after unilateral stapes surgery. In addition, we explore reasons for outpatient failures in otosclerosis surgery. -- Study I examines the outcome of simultaneous bilateral surgery. Hearing was evaluated with standard pure tone and speech audiograms and vestibular apparatus with visual feedback posturography (VFP) during the one-year follow-up. Subjective symptoms and quality of life were assessed with questionnaires. In study II, reasons for outpatient failures in stapes surgery were explored. Forty-seven consecutive stapedotomies and stapedectomies performed by the same surgeon were included, and the effect of failures on hearing results were analysed. Vestibular symptoms and the end organ(s) affected after stapes surgery were investigated in studies III and IV. With video-oculography (VOG), nystagmus was measured preoperatively, and at one week, one month and 3 months postoperatively in the first phase (III). In the second phase (IV), recordings were obtained some hours postoperatively. The hearing results of the simultaneous bilateral surgery were comparable with unilateral surgeries reported. Recovery from the surgery was fast. Significant improvement in performance and quality of life was noted already month after operation in subjective evaluations. Based on these results, simultaneous bilateral surgery is a suitable approach in bilateral otosclerosis Significantly more outpatient failures occurred for medical reasons in the stapedectomy group (13%) than in the stapedotomy group (2%). Stapedotomy should be favoured if outpatient surgery is planned. However, unplanned admission did not worsen the prognosis. VOG measurements in study III did not show any specific type of nystagmus in patients having vestibular symptoms postoperatively. However, VOG measurements immediately after surgery (IV) revealed nystagmus consistent with a minor disturbance of the semicircular canals in 33% of the patients. Subjectively, half of the patients reported vestibular symptoms that were probably of diverse origin, and could have originated from both otolith and semicircular canal parts of the vestibular organ. Since vestibular symptoms and signs are mild, patients may be safely discharged some hours after stapes surgery.

Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Prosopis, an Alien among the Sacred Trees of South India

The problematic of invasive species in an alien environment has aroused the attention of scientists all over the world for quite some time. One of the exotic tree species that has provoked special attention in the tropical drylands is Prosopis juliflora. Originating in South America, prosopis (hereafter referred to as prosopis) has been introduced in the hot and semi-arid zones of the world particularly to provide fuelwood, to stabilize sand dunes and to combat desertification. The tree has become an essential source for fuelwood and a provider of several other products and services in areas where it has become established. However, despite the numerous benefits the tree provides to rural people, in several regions prosopis has become a noxious weed with a negative impact on the environment and to the economy of farmers and landowners. In India, prosopis was introduced in Andhra Pradesh in 1877. The tree was then proclaimed as the precious child of the plant world by scientists and local people alike. The purpose of this study was to investigate the overall impact of prosopis on local rural livelihoods in the drylands of South India. Of particular interest was the examination of the different usages of the tree, especially as fuelwood, and people s perceptions of it. Furthermore, the study examined the negative impacts of the uncontrolled invasion of prosopis on croplands, and its occupation of the banks of irrigation canals and other water sources. As another central theme, this study analysed the Hindu classification system for nature and for trees in particular. In India, several tree species are regarded as sacred. This study examined the position of the exotic prosopis among sacred trees, such as the bodhi, banyan and neem trees. The principle method for collecting the field data was by using individual and thematic group interviews. These interviews were semi-structured with open ended questions. Moreover, unstructured interviews as well as general observations provided complementary information. The data were gathered during two fieldwork periods in the states of Andhra Pradesh and Tamil Nadu, in South India. The results confirmed that prosopis both provides benefits and causes hazards to different stakeholders. Farmers and agriculturalists suffer economic losses in areas where prosopis has invaded crop fields and competes with other plants for water and nutrients. On the other hand, for a significant number of poor rural people, prosopis has become an important source of livelihood benefits. This tree, which grows on government wastelands, is commonly a free resource for all and has thus become a major local source of fuelwood. It also provides several other goods and services and cash income that contributes to improve livelihoods in rural communities. Prosopis ranked lowest in the tree classificatioin system of the Hindus of South India. Although it is appreciated for many benefits it provides for poor people, it has remained an outsider compared with the indigenous tree species. On the other hand, the most sacred trees, such as the bodhi or the banyan, are completely excluded from extraction and it is seen as a sacrilege to even cut branches from any of these trees. An unexpected finding was that, in a few cases, prosopis had also been elevated to the status of a sacred tree. Goods and services from prosopis are not utilized in the most beneficial way. Silvicultural management practices are suggested that would provide additional income and employment opportunities. Interventions are recommended to control further invasion of the tree that might cause serious negative effects in the future. For Hindus, the sacred always ranks highest, even above economic gain. The conservation of sacred groves and sacred trees is a tradition that has its roots in ancient history. These socio-religious practices need to be respected and continued. Successful management of tree and forest resources depends on the willingness of the local people to manage their natural resources, and this willingness exists and has always existed in South India. Keywords: South India, drylands, livelihood, fuelwood, invasive, resource, silviculture.