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- Sloniecka, Marta, 1983-
(författare)
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Neuropeptides and neurotransmitters in keratocytes : importance in corneal wound healing processes
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: The cornea is the outermost transparent layer of the eye and it is responsible for the majorityof the eye’s total focusing power. Keratocytes are the resident cells of the corneal stroma and their function isto produce extracellular matrix components and to take part in corneal healing after injury, which may occurdue to trauma, infection or surgery. The process of corneal wound healing is complex. Shortly, keratocytesadjacent to the corneal wound undergo apoptosis and remaining cells start the process of proliferation andmigration in order to close the wound. Next, an influx of inflammatory cells such as macrophages andneutrophils occurs in order to clear the cornea from cellular debris. The final stage of the healing processrestores the quiescent state of keratocytes and remodels any disordered extracellular matrix components,leading to a healthy, transparent cornea. However, when the process of corneal wound healing is incompleteor disturbed, corneal scarring may occur, which can lead to significantly impaired vision. Despite extensiveresearch on corneal wound healing, corneal scarring remains a major cause of preventable blindness. Thehealing process is dependent on various cytokines and growth factors. However, it is possible that also othersignal substances are involved. Substance P (SP) is a neuropeptide well known for its role in pain perception.It has been shown that SP can also be produced by non-neuronal cells, including cells of the cornea, and thatit can have vast effects on physiological functions, including immune cell activity, and cellular processes, suchas cell migration, proliferation, and production of proinflammatory cytokines. Similarly, acetylcholine (ACh),a classical neurotransmitter, has also been reported to be produced by non-neuronal cells, including cornealepithelium, and to be involved in cell proliferation, angiogenesis, cell migration, apoptosis, and collagen geneexpression. In the studies of this thesis, it is hypothesized that neuropeptides and neurotransmitters areproduced by human keratocytes and that this production is increased in response to corneal injury. Moreover,it is hypothesized that the non-neuronal SP and ACh produced by injured keratocytes participate in cornealwound healing by enhancing keratocyte migration and proliferation, and/or by decreasing keratocyteapoptosis. The aims of this thesis project were to test these hypotheses and to study the underlying inter- andintracellular mechanisms of the effects of SP and ACh on keratocytes.Results: Cultured primary cells of the human corneal stroma expressed keratocyte markers (keratocan,lumican, CD34, and ALDH), the tachykinins SP and NKA, catecholamines (adrenaline, noradrenaline anddopamine), ACh, and glutamate. Moreover, the cells expressed neurokinin-1 and -2 receptors (NK-1R andNK-2R), dopamine receptor D2, muscarinic ACh receptors (mAChRs) M1, M3, M4 and M5, and NDMAR1glutamate receptor. Significant differences were observed between expression profiles in cultured keratocytesobtained from central and peripheral cornea. Such differences could also be seen between keratocytescultured under various serum concentrations. Expression and secretion of SP in cultured keratocytes wasincreased in response to injury in vitro. SP enhanced migration of cultured keratocytes through stimulation ofits preferred receptor, the NK-1R, and activation of the phosphatidylinositide 3-kinase and Rac1/RhoApathway and subsequent actin cytoskeleton reorganization and formation of focal adhesion points. Moreover,SP stimulation led to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8(IL-8), which also contributed significantly to SP-enhanced keratocyte migration and to attractingneutrophils. ACh enhanced keratocyte proliferation in vitro at low concentrations and this stimulation wasmediated through activation of mAChRs and activation of MAPK signalling. Moreover, ACh stimulation led toupregulation of two proliferation markers: PCNA and Ki-67. ACh was also able to protect cultured keratocytesfrom Fas-induced apoptosis, even at low concentrations. Activation of mAChRs was necessary for this latterprocess to occur. ACh reduced caspases 3/7 activation in Fas-treated keratocytes. Inhibition of the PKB/Aktpathway revealed that its activation is essential for mediating the anti-apoptotic effect of ACh in keratocytes.Conclusions: This thesis shows that human keratocytes express an array of neuropeptides (SP, NKA) andneurotransmitters (ACh, adrenaline, noradrenaline, dopamine and glutamate), and their receptors, and thatstimulation of NK-1R by SP and stimulation of mAChRs by ACh lead to keratocyte cellular processes that areknown to be involved in corneal wound healing. Specifically, SP enhances keratocyte migration throughupregulation of IL-8, ACh enhances keratocyte proliferation through activation of the MAPK signallingpathway, and ACh is able to protect keratocytes from apoptosis by activation of the PKB/Akt pathway. Takentogether, these findings suggest that both SP and ACh, if entered at the proper stage, could be beneficial forcorneal wound healing.
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| 2. |
- Spang, Christoph, 1984-
(författare)
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The plantaris tendon in relation to the Achilles tendon in midportion Achilles tendinopathy : studies on morphology, innervation and signalling substances
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Midportion Achilles tendinopathy (tendinosis) is a troublesome painful condition, often characterised by pain, local swelling, tenderness and functional disability. Despite extensive research, the pathogenesis is poorly understood and treatment remains challenging. Features related to the peritendinous connective tissue can be of importance. Recently it has been suggested that the plantaris tendon might be involved in this condition. Furthermore, it has been hypothesised that tendon pain and the tendinosis-related tissue changes in tendinopathy might be mediated by signalling substances such as glutamate and acetylcholine. A clinical observation, not scientifically evaluated, has been that unilateral treatment for bilateral Achilles tendinosis can lead to an effect on the contralateral side. The aim of this work was to examine the morphology and innervation patterns in the plantaris tendon and the peritendinous connective tissue in between the Achillles and plantaris tendons in midportion Achilles tendinopathy, and to evaluate if plantaris tendon removal has an effect on Achilles tendon structure. Another aim was to determine if unilateral treatment for Achilles tendinopathy targeting the peritendinous connective tissue can result in bilateral recovery. Furthermore the presence of non-neuronal cholinergic and glutamate systems was examined. Sections of plantaris tendons with adjacent peritendinous connective tissue from patients with midportion Achilles tendinopathy were stained for morphology (H&E), and innervation patterns were evaluated using antibodies against general nerve marker (PGP9.5), sensory (CGRP) and sympathetic (TH) nerve fibres and Schwann cells (S-100β). Furthermore immunostainings against non-neuronal aceylcholine (ChAT) and glutamate signalling components (glutamate, VGluT2, NMDAR1) were performed. Plantaris tendon cells were cultured and also stained for glutamate signalling components, and were stimulated with glutamate and glutamate receptor agonist NMDA. Furthermore, Ultrasound Tissue Characterisation (UTC) was used to monitor the integrity of the Achilles tendon collagen structure after plantaris tendon removal. Plantaris tendons exhibited tendinosis-like tissue patterns such as hypercellularity, collagen disorganisation and large numbers of blood vessels. The peritendinous connective tissue between the plantaris and Achilles tendons contained large numbers of fibroblasts and blood vessels and to some extent macrophages and mast cells. A marked innervation was found in the peritendinous connective tissue and there were also nerve fibres in the loose connective tissue spaces within the tendon tissue proper. Most nerve fibres were identified as sensory fibres. Some nerve fascicles in the peritendinous connective tissue showed absence of axons but homogenous reactions for Schwann cell marker. Tenocytes and cells in the peritendinous connective tissue expressed ChAT, glutamate, VGluT2 and NMDAR1. Tendon cells in vitro expressed VGluT2, NMDAR1 and glutamate. UTC showed significant improvement of Achilles tendon integrity 6 months after surgical plantaris tendon removal and scraping procedure. Eleven out of thirteen patients reported of a bilateral recovery after unilateral surgical treatment. The results of this work show that plantaris tendons exhibit tendinosis-like tissue changes, internal innervation and features that suggest occurrence of glutamate and acetylcholine production and signalling. Plantaris removal improves Achilles tendon structure suggesting possible compressive/shearing interference between the Achilles and plantaris tendons in tendinopathy. The peritendinous connective tissue shows marked innervation, which thus might transmit pain when being compressed. The partial absence of axons indicates a possible nerve degeneration. On the whole, the study gives new evidence favouring that the plantaris tendon and the peritendinous connective tissue might be of importance for pain and the tendinopathy process in midportion Achilles tendinopathy.
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| 3. |
- Westborg, Inger, 1960-
(författare)
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Safety and efficacy in the cataract surgery process
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- BackgroundCataract and age-related macular degeneration (AMD) are two major causes for visual impairment in the elderly. Cataract surgery is one of the most common surgical interventions performed in the western world. As a consequence of the increasing number of operations performed, postoperative visits are a large workload for surgical units. It is important that all parts of the cataract surgery procedure are appropriate and cost-effective. During the last 20 years the trend is towards fewer visits both pre- as well as postoperatively. The number and timing of postoperative visits are also a subject of an ongoing debate. Few studies have previously evaluated safety perspectives concerning the number and timing of post-operative visits.The last decade, new treatments for wet AMD have evolved and the number of patients receiving treatment has increased. It has been debated if blue-blocking intraocular lenses (IOL) have a protective effect on the development of wet AMD and decreasing the need for AMD-treatment after cataract surgery.AimsTo analyse parts of the modern cataract surgery process including peri- and post-operative routines from a safety and efficacy perspective. To analyse pre- and perioperative risk factors as well as protective factors associated with the need for wet AMD-treatment after cataract surgery.MethodsI, II. These prospective, observational cohort studies included all cataract surgery cases (n=1249) during a 1-year period, at one institution. The cohort was analysed regarding the use of a standardized anaesthetic regimen and the safety perspectives, when the standard routine is no planned postoperative visit in uncomplicated cases without ocular comorbidity.III. The above-mentioned cohort (study group), was compared with a cohort from another clinic (control group) with a different follow-up routine, i.e. each case with first eye cataract surgery had a planned postoperative visit. In the control group all patients (n= 1162 cases) had surgery during the same 1-year period. The number of planned and unplanned visits was recorded, and the surgical outcome from the two institutions was compared.IV. A register-based cohort study included all patients registered in the Swedish National Cataract Register and the Swedish Macula Register in 2010 - 2017, to find all eyes with past cataract surgery that were subsequently treated for wet AMD. Complete registry data was used for comparisons and analyses of pre- and peri-operative risk- and protective factors for wet AMD treatment after cataract surgery.ResultsI. A standardized anaesthetic method with topical and intracameral anaesthetics without sedation was used in most cases (90%). Median pain score after surgery was 0.7 (VAS 0-10) and most patients (97%) would choose the same anaesthetic method again.II, III. Evaluation of all medical records 2 years after the cataract surgery procedure, found no report of missed adverse events. Significantly less patients in the study group (9% vs 16%; p=0.000036) initiated a postoperative unplanned contact compared with the control group. Patients with 70 km or longer to the hospital were less inclined to seek unplanned care (p=0.016).IV. Female gender and high age are associated with an increased risk of needing treatment for wet AMD ≥1 year after cataract surgery. Eyes with a diagnosis of AMD preoperatively, and subsequently treated for wet AMD, had a significantly (p=0.023) lower degree of blue-blocking IOLs implanted at their previous cataract surgery.ConclusionI. A standardized anaesthetic method with topical and intracameral anaesthetics without sedation seems well tolerated by the patients, and is effective at cataract surgery, also in cases when complications/adverse events occur.II, III. Without compromising patient safety, it is possible to refrain from standard postoperative visits after cataract surgery in patients with uncomplicated surgery and no ocular comorbidity. A significant reduction in postoperative visits is only obtained if the standard routine applies to both first and second eye surgery.IV. Patients without preoperative AMD have no benefit from the use of blue-blocking IOLs. In patients with preoperatively diagnosed AMD, blue-blocking IOLs may offer some protection from the subsequent development of AMD.
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