Co-Signaling by Neurotrophic Factors and the Extracellular Matrix for Axonal Growth and Neuronal Survival

Neurotrophic factors (NTFs) and the extracellular matrix (ECM) are important regulators of axonal growth and neuronal survival in mammalian nervous system. Understanding of the mechanisms of this regulation is crucial for the development of posttraumatic therapies and drug intervention in the injured nervous system. NTFs act as soluble, target-derived extracellular regulatory molecules for a wide range of physiological functions including axonal guidance and the regulation of programmed cell death in the nervous system. The ECM determines cell adhesion and regulates multiple physiological functions via short range cell-matrix interactions. The present work focuses on the mechanisms of the action of NTFs and the ECM on axonal growth and survival of cultured sensory neurons from dorsal root ganglia (DRG). We first examined signaling mechanisms of the action of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) on axonal growth. GDNF, neurturin (NRTN) and artemin (ART) but not persephin (PSPN) promoted axonal initiation in cultured DRG neurons from young adult mice. This effect required Src family kinase (SFK) activity. In neurons from GFRalpha2-deficient mice, NRTN did not significantly promote axonal initiation. GDNF and NRTN induced extensive lamellipodia formation on neuronal somata and growth cones. This study suggested that GDNF, NRTN and ARTN may serve as stimulators of nerve regeneration under posttraumatic conditions. Consequently we studied the convergence of signaling pathways induced by NTFs and the ECM molecule laminin in the intracellular signaling network that regulates axonal growth. We demonstrated that co-stimulation of DRG neurons with NTFs (GDNF, NRTN or nerve growth factor (NGF)) and laminin leads to axonal growth that requires activation of SFKs. A different, SFK-independent signaling pathway evoked axonal growth on laminin in the absence of the NTFs. In contrast, axonal branching was regulated by SFKs both in the presence and in the absence of NGF. We proposed and experimentally verified a Boolean model of the signaling network triggered by NTFs and laminin. Our results put forward an approach for predictable, Boolean logics-driven pharmacological manipulation of a complex signaling network. Finally we found that N-syndecan, the receptor for the ECM component HB-GAM was required for the survival of neonatal sensory neurons in vitro. We demonstrated massive cell death of cultured DRG neurons from mice deficient in the N-syndecan gene as compared to wild type controls. Importantly, this cell death could not be prevented by NGF the neurotrophin which activates multiple anti-apoptotic cascades in DRG neurons. The survival deficit was observed during first postnatal week. By contrast, DRG neurons from young adult N-syndecan knock-out mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Corneal nerves in refractive surgery and dry eye

This study aimed to investigate the morphology and function of corneal sensory nerves in 1) patients after corneal refractive surgery and 2) patients with dry eye due to Sjögren's syndrome. A third aim was to explore the possible correlation between cytokines detected in tears and development of post-PRK subepithelial haze. The main methods used were tear fluid ELISA analysis, corneal in vivo confocal microscopy, and noncontact esthesiometry. The results revealed that after PRK a positive correlation exists between the regeneration of subbasal nerves and the thickness of regenerated epithelium. Pre- or postoperative levels of the tear fluid cytokines TGF-β1, TNF-α, or PDGF-BB did not correlate with the development of corneal haze objectively estimated by in vivo confocal microscopy 3 months after PRK. After high myopic LASIK, a discrepancy between subjective dry eye symptoms and objective signs of dry eye was observed. The majority of patients reported ongoing dry eye symptoms even 5 years after LASIK, although no objective clinical signs of dry eye were apparent. In addition, no difference in corneal sensitivity was observed between these patients and controls. Primary Sjögren's syndrome patients presented with corneal hypersensitivity, although their corneal subbasal nerve density was normal. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased number of antigen-presenting cells among subbasal nerves were observed, implicating the presence of an ongoing inflammation. Based on these results, the relationship between nerve regeneration and epithelial thickness 3 months after PRK appears to reflect the trophic effect of corneal nerves on epithelium. In addition, measurement of tear fluid cytokines may not be suitable for screening patients for risk of scar (haze) formation after PRK. Presumably, at least part of the symptoms of "LASIK-associated dry eye" are derived from aberrantly regenerated and abnormally functioning corneal nerves. Thus, they may represent a form of corneal neuropathy or "phantom pain" rather than conventional dry eye. Corneal nerve alterations and inflammatory findings in Sjögren's syndrome offer an explanation for the corneal hypersensitivity or even chronic pain or hyperalgesia often observed in these patients. In severe cases of disabling chronic pain in patients with dry eye or after LASIK, when conventional therapeutic possibilities fail to offer relief, consultation of a physician specialized in pain treatment is recommended.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Natural regeneration and ecological succession in Pinus kesiya watershed plantations in northern Thailand : Implications on plantation management

Establishment of Pinus kesiya Roy. ex Gord. plantations in Thailand began in the 1960s by the Royal Forest Department. The aim was to reforest abandoned swidden areas and grasslands in order to reduce erosion and to produce timber and fuel wood. Today there are about 150, 000 ha of P. kesiya plantations in northern Thailand. Most of these plantations cannot be harvested due to a national logging ban. Previous studies have suggested that Pinus kesiya plantations posses a capability as a foster environment for native broadleaved tree species, but little is known about the extent of regeneration in these plantations. The general aim of the study was to clarify the extent of forest regeneration and interactions behind it in Pinus kesiya plantations of the Ping River basin, northern Thailand. Based on the results of this study and previous literature, forest management proposals were produced for the area studied. In four different pine plantation areas, a total of seven plantations were assessed using systematic data collection with clustered circular sample plots. Vegetation and environmental data were statistically analysed, so as to recognise the key factors affecting regeneration. Regeneration had occurred in all plantations studied. Regeneration of broadleaved trees was negatively affected by forest fire and canopy coverage. A high basal area of mature broadleaved trees affected the regeneration process positively. Forest fire disturbance had a strong effect also on plantation structure and species composition. Because of an unclear future forest management setting as regards forest laws in Thailand, a management system that enables various future utilisation possibilities and emphasises local participation is recommended for P. kesiya watershed platations of northern Thailand.

The visual preferences for forest regeneration and field afforestation - four case studies in Finland

The overall aim of this dissertation was to study the public's preferences for forest regeneration fellings and field afforestations, as well as to find out the relations of these preferences to landscape management instructions, to ecological healthiness, and to the contemporary theories for predicting landscape preferences. This dissertation includes four case studies in Finland, each based on the visualization of management options and surveys. Guidelines for improving the visual quality of forest regeneration and field afforestation are given based on the case studies. The results show that forest regeneration can be connected to positive images and memories when the regeneration area is small and some time has passed since the felling. Preferences may not depend only on the management alternative itself but also on the viewing distance, viewing point, and the scene in which the management options are implemented. The current Finnish forest landscape management guidelines as well as the ecological healthiness of the studied options are to a large extent compatible with the public's preferences. However, there are some discrepancies. For example, the landscape management instructions as well as ecological hypotheses suggest that the retention trees need to be left in groups, whereas people usually prefer individually located retention trees to those trees in groups. Information and psycho-evolutionary theories provide some possible explanations for people's preferences for forest regeneration and field afforestation, but the results cannot be consistently explained by these theories. The preferences of the different stakeholder groups were very similar. However, the preference ratings of the groups that make their living from forest - forest owners and forest professionals - slightly differed from those of the others. These results provide support for the assumptions that preferences are largely consistent at least within one nation, but that knowledge and a reference group may also influence preferences.

Corneal recovery after uncomplicated and complicated PRK and LASIK

Refractive errors, especially myopia, seem to increase worldwide. Concurrently, the number of surgical refractive corrections has increased rapidly, with several million procedures performed annually. However, excimer laser surgery was introduced after a limited number of studies done with animals and to date there still are only few long-term follow-up studies of the results. The present thesis aims to evaluate the safety and functional outcome of, as well as to quantify the cellular changes and remodelling in the human cornea after, photorefractive keratectomy (PRK) and laser assisted in situ keratomileusis (LASIK). These procedures are the two most common laser surgical refractive methods. In Study I, myopic ophthalmic residents at Helsinki University Eye Hospital underwent a refractive correction by PRK. Five patients were followed up for 6 months to assess their subjective experience in hospital work and their performance in car driving simulator and in other visuomotor functions. Corneal morphological changes were assessed by in vivo confocal microscopy (ivCM). Study II comprised 14 patients who had undergone a PRK operation in 1993-1994. Visual acuity was examined and ivCM examinations performed 5 years postoperatively. In Study III 15 patients received LASIK refractive correction for moderate to high myopia (-6 - -12 D). Their corneal recovery was followed by ivCM for 2 years. Diffuse lamellar keratitis (DLK) is a common but variable complication of LASIK. Yet, its aetiology remains unknown. In Study IV we examined six patients who had developed DLK as a consequence of formation of an intraoperative or post-LASIK epithelial defect, to assess the corneal and conjunctival inflammatory reaction. In the whole series, the mean refractive correction was -6.46 diopters. The best spectacle corrected visual acuity (BSCVA) improved in 30 % of patients, whereas in four patients BSCVA decreased slightly. The mean achieved refraction was 0.35 D undercorrected. After PRK, the stromal scar formation was highest at 2 to 3 months postoperatively and subsequently decreased. At 5 years increased reflectivity in the subepithelial stroma was observed in all patients. Interestingly, no Bowman s layer was detected in any patient. Subbasal nerve fiber bundle(snfb) regeneration could be observed already at 2 months in 2 patients after PRK. After 5 years, all corneas presented with snfb, the density of which, however, was still lower than in control corneas. LASIK induced a hypocellular area on both sides of the flap interface. A decrease of the most anterior keratocyte density was also observed. In the corneas that developed DLK, inflammatory cell-type objects were present in the flap interface in half of the patients. The other patients presented only with keratocyte activation and highly reflective extracellular matrix. These changes resolved completely with medication and time. Snfb regeneration was first detected at one month post-LASIK, but still after two years the density of snfb, however, was only 64 % of the preoperative values. The performance of ophthalmological examinations and microsurgery without spectacles was easier postoperatively, which was appreciated by the residents. Both PRK and LASIK showed moderate to good accuracy and high safety. In terms of visual perception and subjective evaluation, few patients stated any complaints in the whole series of studies. Instead, the majority of patients experienced a marked improvement in everyday life and work performance. PRK and LASIK have shown similar results, with good long term morphological healing. It seems evident that, even without the benefit of over-20-year follow-up results, these procedures are sufficiently safe and accurate for refractive corrections and corneal reshaping.

Regeneration ecology and woody species diversity in the remnant natural forest at Wondo Genet, Ethiopia – Potential for landscape restoration

Regeneration ecology, diversity of native woody species and its potential for landscape restoration was studied in the remnant natural forest at the College of Forestry and Natural Resources at Wondo Genet, Ethiopia. The type of forest is Afromontane rainforest , with many valuable tree species like Aningeria adolfi-friederici, and it is an important provider of ecological, social and economical services for the population that lives in this area. The study contains two parts, natural regeneration studies (at the natural forest) and interviews with farmers in the nearby village of the remnant patch. The objective of the first part was to investigate the floristic composition, densitiy and regeneration profiles of native woody species in the forest, paying special attention to woody species that are considered the most relevant (socio-economic). The second part provided information on woody species preferred by the farmers and on multiple uses of the adjacent natural forest, it also provided information and analysed perceptions on forest degradation. Systematic plot sampling was used in the forest inventory. Twenty square plots of 20 x 20 m were assessed, with 38 identified woody species (the total number of species was 45), representing 26 families. Of these species 61% were trees, 13% shrubs, 11% lianas and 16% species that could have both life forms. An analysis of natural regeneration of five important tree species in the natural forest showed that Aningeria adolfi-friederici had the best regeneration results. An analysis of population structure (as determined by height classes) of two commercially important woody species in the forest, Aningeria adolfi-friederici and Podocarpus falcatus, showed a marked difference: Aningeria had a typical “reversed J” frequency distribution, while Podocarpus showed very low values in all height classes. Multi dimensional scaling (MDS) was used to map the sample plots according to their similarity in species composition, using the Sørensen quantitative index, coupled with indicator species analysis .Three groups were identified with respective indicator species: Group 1 – Adhatoda schimperiana, Group 2 – Olea hochstetteri , Group 3 – Acacia senegal and Aningeria adolfi-friederici. Thirty questionnaire interviews were conducted with farmers in the village of Gotu Onoma that use the nearby remant forest patch. Their tree preferences were exotic species such as Eucalyptus globulus for construction and fuelwood and Grevillea robusta for shade and fertility. Considering forest land degradation farmers were aware of the problem and suggested that the governmental institutions address the problem by planting more Eucalyptus globulus. The natural forest seemed to have moderate levels of disturbance and it was still floristically diverse. However, the low rate of natural regeneration of Podocarpus falcatus suggested that this species is threatened and must be a priority in conservation actions. Plantations and agroforestry seem to be possible solutions for rehabilitation of the surrounding degraded lands, thereby decreasing the existent pressure in the remnant natural forest.

Experimental approach for studying structural changes in axonal membrane upon nerve excitation

The Hodgkin and Huxley (HH) model of action potential has become a central paradigm of neuroscience. Despite its ability to predict action potentials with remarkable accuracy, it fails to explain several biophysical findings related to the initiation and propagation of the nerve impulse. The isentropic heat release and optical phenomena demonstrated by various experiments suggest that action potential is accompanied by a transient phase change in the axonal membrane. In this study a method was developed for preparing a giant axon from the crayfish abdominal cord for studying the molecular mechanisms of action potential simultaneously by electrophysiological and optical methods. Also an alternative setup using a single-cell culture of an Aplysia sensory neuron is presented. In addition to the description of the method, the preliminary results on the effect of phloretin, a dipole potential lowering compound, on the excitability of a crayfish giant axon are presented.

The impact of FSC certification on timber tree regeneration and floristic composition in Honduran community forests

Forest certification has been put forward as a means to improve the sustainability of forest management in the tropical countries, where traditional environmental regulation has been inefficient in controlling forest degradation and deforestation. In these countries, the role of communities as managers of the forest resources is rapidly increasing. However, only a fraction of tropical community forests have been certified and little is known about the impacts of certification in these systems. Two areas in Honduras where community-managed forest operations had received FSC certifications were studied. Río Cangrejal represents an area with a longer history of use, whereas Copén is a more recent forest operation. Ecological sustainability was assessed through comparing timber tree regeneration and floristic composition between certified, conventionally managed and natural forests. Data on woody vegetation and environmental conditions was collected within logging gaps and natural treefall gaps. The regeneration success of shade-tolerant timber tree species was lower in certified than in conventionally managed forests in Río Cangrejal. Furthermore, the floristic composition was more natural-like in the conventionally managed than the certified forests. However, the environmental conditions indicated reduced logging disturbance in the certified forests. Data from Copén demonstrated that the regeneration success of light-demanding timber species was higher in the certified than the unlogged forests. In spite of this, the most valuable timber species Swietenia macrophylla was not regenerating successfully in the certified forests, due to rapid gap closure. The results indicate that pre-certification loggings and forest fragmentation may have a stronger impact on forest regeneration than current, certified management practices. The focus in community forests under low-intensive logging should be directed toward landscape connectivity and the restoration of degraded timber species, instead of reducing mechanical logging damage. Such actions are dependent on better recognition of resource rights, and improving the status of small Southern producers in the markets of certified wood products.

Effect of soil preparation of boreal spruce forest on air and soil temperature conditions in forest regeneration areas.

The effect of scarification, ploughing and cross-directional plouhing on temperature conditions in the soil and adjacent air layer have been studied during 11 consecutive growth periods by using an unprepared clear-cut area as a control site. The maximum and minimum temperatures were measured daily in the summer months, and other temperature observations were made at four-hour intervals by means of a Grant measuring instrument. The development of the seedling stand was also followed in order to determine its shading effect on the soil surface. Soil preparation decreased the daily temperature amplitude of the air at the height of 10 cm. The maximum temperatures on sunny days were lower in the tilts of the ploughed and in the humps of the cross-directional ploughed sites compared with the unprepared area. Correspondingly, the night temperatures were higher and so the soil preparation considerably reduced the risk of night frost. In the soil at the depth of 5 cm, soil preparation increased daytime temperatures and reduced night temperatures compared with unprepared area. The maximum increase in monthly mean temperatures was almost 5 °C, and the daily variation in the surface parts of the tilts and humps increased so that excessively high temperatures for the optimal growth of the root system were measured from time to time. The temperature also rose at the depths of 50 and 100 cm. Soil preparation also increased the cumulative temperature sum. The highest sums accumulated during the summer months were recorded at the depth of 5 cm in the humps of cross-directional ploughed area (1127 dd.) and in the tilts of the ploughed area (1106 dd.), while the corresponding figure in the unprepared soil was 718 dd. At the height of 10 cm the highest temperature sum was 1020 dd. in the hump, the corresponding figure in the unprepared area being 925 dd. The incidence of high temperature amplitudes and percentage of high temperatures at the depth of 5 cm decreased most rapidly in the humps of cross-directional ploughed area and in the ploughing tilts towards the end of the measurement period. The decrease was attributed principally to the compressing of tilts, the ground vegetation succession and the growth of seedlings. The mean summer temperature in the unprepared area was lower than in the prepared area and the difference did not diminish during the period studied. The increase in temperature brought about by soil preparation thus lasts at least more than 10 years.

Microsite occupancy and spatial structure of regeneration in three late-successional Norway spruce forests in northern Europe

A comparison of microsite occupancy and the spatial structure of regeneration in three areas of late-successional Norway spruce dominated forest. Pallas-Ylläs is understood to have been influenced only by small-scale disturbance; Dvina-Pinega has had sporadic larger-scale disturbances; Kazkim has been affected by fire. All spruce and birch trees with diameter at breast height (DBH) ?10 cm were mapped in five stands on 40 m x 400 m transects, and those with DBH < 10 cm on 2 or 4 m x 400 m subplots. Microsite type was inventoried at 1m intervals along the centre line and for each tree with DBH < 10 cm. At all study areas small seedlings (h < 0.3 m, DBH < 10 cm) preferentially occupied disturbed microsites. In contrast, spruce saplings (h ? 1.3 m, DBH <10 cm) at all study areas showed less, or no, preference. At Pallas-Ylläs spruce seedlings (h < 1.3 m, DBH < 10 cm) and saplings (h ? 1.3 m, DBH < 10 cm) exhibited spatial correlation at scales from 32-52 m. At Dvina-Pinega saplings of both spruce and birch exhibited spatial correlation at scales from 32-81 m. At Kazkim spatial correlation of seedlings and saplings of both species was exhibited over variable distances. No spatial cross-correlation was found between overstorey basal area (DBH ? 10 cm) and regeneration (h ? 1.3 m, DBH < 10 cm) at any study area. The results confirm the importance of disturbed microsites for seedling establishment, but suggest that undisturbed microsites may sometimes be more advantageous for long-term tree survival. The regeneration gap concept may not be useful in describing the regeneration dynamics of late-successional boreal forests.