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The effects of plasminogen deficiency on the healing of tympanic membrane perforations
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The healing of tympanic membrane (TM) perforations is a complex wound healing process including inflammation, migration of keratinocytes and tissue remodelling. Most TM perforations in human heal spontaneously, however some perforations become chronic, and the reason to why is still largely unknown. In cutaneous wound healing plasminogen (plg) has been shown to play an important role. Plg is converted into the protease plasmin regulated by two plasminogen activators (PA), urokinase type PA (uPA) and tissue-type PA (tPA).

The aim of the present thesis was to evaluate the role of plg in healing of TM perforations, both in vivo and in vitro. The main objectives were to determine the healing capacity of the TM, the involvement of keratinocytes, fibrin(ogen) and inflammatory cells in the healing process. The studies were performed in plg deficient and uPA deficient mice, with littermate wild type (wt) mice as controls

It was shown that myringotomies of the TMs in plg deficient mice still remained open 143 days following a perforation. The wound area was characterized by an abundant recruitment and accumulation of inflammatory cells; mainly macrophages and neutrophils, an arrested keratinocyte migration and a fibrin deposition covering the surface of the TM. The TM perforations in the wt mice all healed within 11 days. Interestingly, the myringotomies of the plg deficient mice could be closed by reconstitution with systemic injections of plg, whereas injections of PBS had no affect on the healing.

To characterize mechanisms involved in the development of persistent TM perforations in plg deficient mice after a myringotomy the early inflammatory response during the first 48 hours was studied. The recruitment and accumulation of inflammatory cells in the perforated TMs was found to be similar between the plg deficient and the wt mice.

Myringotomized TMs in uPA deficient mice healed similar to perforations of wt controls. Neither did the keratinocyte migration nor the occurrence of inflammatory cells differ between these genotypes.

In the in vitro experiments TMs from plg deficient and wt mice, were dissected out, perforated and cultured in absence or surplus of plg. A decrease in perforation size was seen in all groups regardless of genotype or amount of plg in the medium.

In conclusion, the present studies show:

• Plg is essential for the healing of TM perforations in mice.

• The altered healing process after a myringotomy in plg deficient mice involves a disturbed keratinocyte migration, a massive deposition of fibrin and an abundant accumulation of inflammatory cells in the wound area.

• Plasminogen deficiency does not alter the early inflammatory response, following a myringotomy.

• Deficiency of uPA does not influence the healing of TM perforations.

• During in vitro conditions healing of TM perforations is initiated irrespectively of genotype of the explant (plg deficient or wt) or supply of plg.

The increased knowledge of the involvement of plg in the healing of TM perforations may open therapeutical possibilities in the treatment of chronic TM perforations in humans.

Place, publisher, year, edition, pages
Umeå: Klinisk vetenskap , 2007. , 66 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1090
Keyword [en]
tympanic membrane, macrophages, neutrophils, fibrin(ogen), inflammation, keratinocyte migration, wound healing, uPA, tPA, in vitro
National Category
Otorhinolaryngology
Identifiers
URN: urn:nbn:se:umu:diva-1100ISBN: 978-91-7264-276-8 (print)OAI: oai:DiVA.org:umu-1100DiVA: diva2:140199
Public defence
2007-05-18, Sal D, Tandläkarhögskolan (Dallashuset), Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-04-27 Created: 2007-04-27 Last updated: 2011-04-08Bibliographically approved
List of papers
1. Plasmin/plasminogen is essential for the healing of tympanic membrane perforations.
Open this publication in new window or tab >>Plasmin/plasminogen is essential for the healing of tympanic membrane perforations.
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2006 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 96, no 4, 512-519 p.Article in journal (Refereed) Published
Abstract [en]

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Keyword
Animals, Cell Proliferation, Fibrin/metabolism, Keratinocytes/metabolism/pathology, Keratins/metabolism, Macrophages/immunology, Male, Mice, Mice; Inbred C57BL, Mice; Inbred DBA, Mice; Knockout, Neutrophil Infiltration, Neutrophils/immunology, Plasmin/*metabolism, Plasminogen/genetics/*metabolism/pharmacology, Time Factors, Tympanic Membrane Perforation/immunology/*metabolism/pathology, Wound Healing/drug effects
Identifiers
urn:nbn:se:umu:diva-6327 (URN)10.1160/TH06-03-0168 (DOI)17003931 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2011-03-18Bibliographically approved
2. Lack of plasminogen does not alter the early inflammatory response following a tympanic membrane perforation: a study in plasminogen-deficient mice.
Open this publication in new window or tab >>Lack of plasminogen does not alter the early inflammatory response following a tympanic membrane perforation: a study in plasminogen-deficient mice.
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2008 (English)In: Acta Oto-Laryngologica, ISSN 0001-6489, E-ISSN 1651-2251, Vol. 128, no 12, 1294-1302 p.Article in journal (Refereed) Published
Abstract [en]

CONCLUSIONS: The results of the present study show that the early inflammatory response in plasminogen (plg)-deficient mice is not altered compared to that in wild-type (wt) mice. Therefore the chronicity of the perforation in the long-term healing experiment cannot be explained by an impairment of the early inflammatory response, but rather by an impairment in activation of the inflammatory cells. These findings give further insight into the mechanisms resulting in a clinically seen chronic tympanic membrane (TM) perforation and thus possible therapeutic strategies to replace today's conventional surgical treatment of these perforations. OBJECTIVES: Plg has been shown to play an essential role in the healing of TM perforations. In plg-deficient mice a completely arrested healing reaction was seen, resulting in a chronic TM perforation. The mechanisms involved seem to be an abundant neutrophil recruitment, an accumulation of macrophages, an arrested keratinocyte migration, and a massive deposition of fibrin along the TM tissue. However, the exact functional role of plg in the early inflammatory response during healing of TM perforation remains unclear. This study aimed to evaluate the early inflammatory response, mainly the occurrence of macrophages and neutrophils, during the first 48 h following a perforation in the pars tensa (PT) of the TM, in mice lacking the plasminogen gene compared to the corresponding response in wt mice. MATERIALS AND METHODS: The TMs were perforated in 45 plg-deficient and 39 wt mice. Otomicroscopic evaluation was performed at 3, 6, 9, 12, 18, 24, and 48 h after the perforation was made. Mice were harvested at all time points and prepared for morphology including immunohistochemistry (IHC). IHC was performed with antibodies targeting macrophages, neutrophils, T and B cells, cytokeratin, and fibrin(ogen). Morphometry was performed regarding the volume percentage of TM tissue occupied by the different inflammatory cells. RESULTS: Perforation of the TM resulted in early otomicroscopic changes of the pars flaccida (PF) in both genotypes. Infiltration of inflammatory cells to PF and the presence of edema occurred as early as 6 h after the perforation was made, in both plg-deficient and wt mice. Morphometry did not reveal any significant differences between the genotypes concerning the occurrence of inflammatory cells. In contrast to the PF, the PT showed only sparse reactions during the experimental period. Furthermore, the migration pattern of keratinocytes did not differ between the genotypes throughout the experimental period.

Identifiers
urn:nbn:se:umu:diva-18629 (URN)10.1080/00016480701361996 (DOI)18781446 (PubMedID)
Available from: 2009-02-19 Created: 2009-02-19 Last updated: 2017-12-13
3. The healing of tympanic membrane perforations is unaffected by urokinase-type plasminogen activator deficiency
Open this publication in new window or tab >>The healing of tympanic membrane perforations is unaffected by urokinase-type plasminogen activator deficiency
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2271 (URN)
Available from: 2007-04-27 Created: 2007-04-27 Last updated: 2011-03-30Bibliographically approved
4. Plasminogen deficiency does not alter the healing of tympanic membrane perforations in vitro
Open this publication in new window or tab >>Plasminogen deficiency does not alter the healing of tympanic membrane perforations in vitro
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(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2272 (URN)
Available from: 2007-04-27 Created: 2007-04-27 Last updated: 2011-03-30Bibliographically approved

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