Although the classical method of normalization of a single housekeeping gene is assumed to be invariable among different tissue samples, their individual expression may vary with respect to different experimental conditions, disease state, neoplasia, age of the animal and the tissue being analyzed, hence, influencing the correct interpretation of results. To circumvent such issues, high density endogenous control array pre-designed with primers and probes for 16 housekeeping genes was preferred over the normalization against a single gene. The modiolus contains the cell bodies of the SGC and auditory nerve (Stoever et al., 2001). The current study focused on the gene expression pattern of GDNF, GFRα-1, BDNF, TrkB, p75NTR, bcl2, bax, caspase-9 and -3 mRNA in the
modiolus using real-time PCR and localized these proteins to the SGC of the auditory nerve in the cochlea using immunohistochemistry. For the estimation of RNA quality in the modiolus, quality control from Agilent bioanalyzer was performed. A RNA integrity number (RIN) of more than 7 was found in all the tissue pools constituting normal hearing (NH), contralateral and deafened modiolus, indicating that the RNA extracted from these tissue pools were intact and not degraded. The highly specific Taqman gene expression assays (“m1”) used in the study spanning the exon junction may exclude gene products with similar homologs and off-target sequences, and are not affected by genomic DNA contamination. Lastly, the amplification efficiency regarding the potential contamination of modiolus from the surrounding mixed tissues has to be considered.
Nevertheless, our statistical significant gene expression results shows that the mere contaminants present in the modiolus may not influence the changes at the gene expression levels.
Determination of apoptosis and cell survival signaling following neomycin induced deafness in the rat cochlea
Summary
Aminoglycoside-induced sensorineural hearing loss (SNHL) is associated with loss of hair cells followed by secondary degeneration of spiral ganglion cell (SGC). By the common “neurotrophin hypothesis”, cell death associated with degeneration of neurons may reflect deprivation of neurotrophic factors. Contrary to this, recent studies have reported that gene expression profiling of neurotrophic factors, GDNF, BDNF, artemin, GFRα-1 and transforming growth factor beta 1 and 2 (TGF1/2) were significantly upregulated following 26 days of neomycin-induced deafness in the rat cochleae. The reasons for the contradictory findings may be as follows: (i) it is well known that some growth factors or their receptors both contribute to the cell survival and protection as well as cell death. However, it is still unclear in which way they shift the delicate balance of survival and cell death, (ii) The autocrine neurotrophic factor expression may be induced at a time point the degeneration process can only be slowed down, (iii) overexpression of the neurotrophic factors may not be able to ensure the SGC survival throughout a longer period following the neomycin induced degeneration., thus, additional external application of neurotrophic factors may be needed.
In consideration of the neurotrophin hypothesis and the previous findings in gene expression analysis following 26 days deafness, this study presents the results of SGC survival and gene expression profiling of GDNF, BDNF, their corresponding receptors GFR1, TrkB and p75NTR, pro- and anti-apoptotic molecules in the rat cochleae following 7, 14 and 28 days deafening. Hereby, we suppose, that the ongoing of the SGC degeneration despite the strong neurotrophic factor upregulation might be the result of deprivation induced induction of the neurotrophic factor expression.
Souvik Kar
Hearing sensitivity was confirmed by frequency specific acoustic auditory brainstem responses (AABR) and SGC loss density was estimated morphometrically by cell counts to determine the extent of degeneration in the Rosenthal’s canal at different time periods following deafening. As shown by immunohistochemical staining the expression of GDNF, BDNF, GFRα-1, TrkB, p75NTR, bcl-2, bax, caspase-9 and caspase-3 was positive to SGC. Differential gene expression patterns of GDNF, GFRα-1, BDNF, p75NTR and caspase-3 were significantly upregulated following 28 days of deafening. We observed a slight upregulation of pro-apoptotic molecule bax at 14 day and caspase-9 at 28 days in the deafened modiolus, respectively. There were no statistical significant differences in the expression level in the contralateral modioli at days 7 and 14 post deafening. In contrast, a reciprocal expression of TrkB and p75NTR was noticed, in which p75NTR was upregulated and TrkB was downregulated in the deaf modiolus. These findings demonstrated that neomycin induced a high and low-frequency hearing loss in the deafened and contralateral ear, respectively. SGC cell counts confirmed a significant loss of neurons in a time dependent manner. As revealed by the data from gene expression analysis, substantial increase of GDNF and its receptor GFRα-1 following deafening indicated deprivation-induced upregulation in the SGC after hair cell loss. The degenerating SGC are maintained to survive for a longer time period by the Schwann cells, which remain a potential source of neurotrophic support. Deafened animals exhibiting higher expression levels of BDNF and p75NTR and reduced TrkB mRNA, indicated a reciprocal cross-talk following deafening. We speculated that p75NTR binds to BDNF and triggers apoptosis in neurons resulting in the degeneratuin process. This hypothesis may be supported by the upregulation of bax and caspase-3 in the deafened modiolus. We assume that an alternate cell-death pathway is induced in our neomycin-induced deafness model that may trigger such phenomenon. To summarize, the differential gene expression changes of GDNF and its receptor GFRα-1, BDNF, p75NTR and TrkB indicate a fine-tuned orchestration between cell survival and death in this neomycin-deafness model. Moreover, these changes in the expression levels following deafening are in consistence with the ‘neurotrophin hypothesis’ by which apoptosis of the SGC death is a result of deprivation of neurotrophic factors not only supplied by the Schwann cells. Understanding the response of Schwann cells to deafening might facilitate
SGC maintenance and regeneration studies. Future studies should aim to investigate extended time period of deafness and try to understand the similarities and differences between ototoxic mechanisms underlying SNHL, in order to develop more protective strategies to minimize their ototoxicity.
Untersuchung der Signalwege der Apoptose und des Zellüberlebens nach Ertaubung durch Applikation von Neomycin in die Rattencochlea
Zusammenfassung
Innenohrschwerhörigkeit ist mit dem Verlust der Haarzellen durch sekundäre Degeneration der Spiralganglion-Neuronen (SGN) nach einem Ototrauma verbunden.
Nach der „Neurotrophin-Hypothese“ erfolgt die Degeneration der Hörnerven nach Haarzellverlust durch Unterbrechung/Entzug der trophischen Aktivität neurotropher Faktoren (NTF) als Folge der von sensorischen Zellen induzierten Apoptose. Gemäß den Ergebnissen der Genexpressionsanalyse der bisher untersuchten Mitglieder der GDNF-Familie und TGF-Superfamilie, die als sog. „survival“-Faktoren vor den Folgen ototoxischen Traumas im Innerohr von Nagetieren charakterisiert sind, läßt sich die oben genannte Hypothese nicht aufrechterhalten: Die Transkriptionsrate von GDNF, artemin und BDNF wurde im Hörnerven erwachsener Ratten 26 Tage nach der Ertaubung durch Neomycin entgegen dem Postulat der „Neurotrophin-Hypothese“ erhöht. Folgende Gründe für die erhöhte Expression könnten eine Rolle spielen: (i) Es ist bekannt, dass einige Wachstumsfaktoren sowohl zur Zellprotektion beitragen als auch die Apoptose fördern können. Unklar ist, wann und bei welchen externen und/oder intrazellulären Stimuli sich die Funktionen umkehren. (ii) Die autokrine Expression der NTF wird zu einem späteren Zeitpunkt induziert und verlangsamt den Degenerationsprozeß. (iii) Das Überleben der SGN nach einem aminoglykosid-induzierten Degenerationsprozess für einen längeren Zeitraum ist nicht ausreichend, so dass zusätzlich NTF von außen appliziert werden muss.
Basierend auf der Neurotrophin-Hypothese und den Ergebnissen der Genexpressionstudie in ertaubten Ratten nach 26 Tagen untersucht die vorliegende Studie die Expression von NTF im Rahmen der Induktion apoptotischer Signale in den Modioli der Ratten-Cochleae 7, 14 und 28 Tage nach der Neomycin induzierten Ertaubung auf mRNA- und
Souvik Kar
Proteinebene. Der Hörstatus der Versuchstiere wurde elektrophysiologisch mittels
„acoustically evoked auditory brainstem response“ (AABR) vor und 7, 14 und 28 Tage nach der Ertaubung bestimmt. Die differentielle Genexpression von GDNF und BDNF, deren korrespondierenden Rezeptoren GFRα-1, p75NTR und Trk-B, Bcl-2 (antiapoptotisches Signalmolekül), Bax, Caspasen 3 und 9 (apoptitische Signalmoleküle) wurden mittels real time-PCR untersucht. Zur Bestimmung der Ausdehnung der SGN-Degeneration zu den oben angegebenen Zeitpunkten nach der Neomycin-Injektion wurde die Dichte der SGN histologisch durch Auszählen noch intakter Soma der SGN in den Rosenthal-Kanälen ermittelt. Die subzelluläre Lokalisation von neurotrophen und apoptotischen Markern auf Proteinebene wurde immunhistochemisch bestimmt.
Immunhistochemisch wurde die Expression von GDNF, BDNF, GFRα-1, TrkB, p75NTR, bcl-2, bax, caspase-9 and caspase-3 im Spiralganglion nachgewiesen.
Die Analyse der Transkriptionsaktivität im Modiolus 28 Tage nach der Ertaubung zeigte eine signifikante Hochregulierung von GDNF und dessen Rezeptor GFRα-1, BDNF und dessen Rezeptor p75NTR sowie Caspase-3 im Vergleich zu den normal hörenden (NH) Tieren. Im Gegensatz dazu wurde die Genexpression des BDNF-spezifischen Rezeptors TrkB im Vergleich zur NH-Gruppe am Tag 28 signifikant herunterreguliert. Der mRNA-Level des proapoptotischen Signalmoleküls Bax war nur am Tag 14 der Ertaubung signifikant erhöht, während an Tag 28 die Transkriptionsaktivität wiederum herunterreguliert wurde. Im Vergleich zur NH-Gruppe wurde ebenfalls nur 28 Tage nach der Ertaubung eine signifikante Erhöhung der Caspase-3-Genexpression nachgewiesen.
Dagegen wurden keine signifikanten Änderungen der Caspase-9-Genexpression an allen Untersuchungstagen gefunden. Für die Bcl2-Genexpression wurde eine leichte, aber nichtsignifikante, Herunterregulierung 7, 14 und 28 Tage nach der Ertaubung ermittelt.
Keine statistisch signifikanten Änderungen im Expressionslevels wurden in den kontralateralen Ohren der Versuchstiere gefunden, die für 7 und 14 Tage ertaubt waren.
Unsere Daten in der Bestimmung der SGN-Dichte bestätigten einen signifikanten Verlust der SGN mit zunehmender Zeitdauer der Ertaubung. Darüber hinaus sprechen die Aktivierung von Bax, Caspase-9 und -3 sowie die verringerte Genexpressionsaktivität des antiapoptotischen Bcl-2 für eine caspaseinduzierte mitochondriale Apoptose in ertaubten Ratten. Im Zusammenhang mit dem Anstieg der Genexpression von BDNF und
p75NTR sowie der Herunterregulierung der TrkB-Genexpression ist ein reziproker „cross-talk“ im Degenerationsprozeß der SGN zu vermuten: Durch Bindung von BDNF an p75NTR wurde möglicherweise die Apoptose in den Neuronen getriggert. Die Hochregulierung von bax und caspase-3-mRNA unterstützt diese Annahme.
Möglicherweise stellen hauptsächlich Schwannzellen die Quelle für die erhöhten Level der GDNF- und GFR-1-mRNA dar und die autokrine Expression der NTF als „survival Faktoren“ in den SGN spielt selbst keine Rolle mehr. Nach dieser Schlußfolgerung kann das Postulat der Neurotrophin-Hypothese als erfüllt betrachtet werden. Welche Rolle Schwannzellen im Degenerationsprozeß und dem Erhalt der SGN spielt, wird weiterhin Gegenstand der Innenohrforschung sein.
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