Basic Science

Graefe's Archive for Clinical and Experimental Ophthalmology

, Volume 252, Issue 2, pp 267-274

First online:

Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens

  • Alessandra MiceraAffiliated withIRCCS-G.B. Bietti FoundationLaboratory of Ophtalmology, IRCCS-G.B. Bietti Foundation Email author 
  • , Katerina JirsovaAffiliated withLaboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital
  • , Eduardo Maria NormandoAffiliated withCIR, Department of Ophthalmology, University Campus Bio-MedicoGlaucoma & Retinal Degeneration Research Group, Visual Neurosciences, UCL Institute of OphthalmologyThe Western Eye Hospital, Imperial College Healthcare Trust
  • , Barbara StampachiacchiereAffiliated withIRCCS-G.B. Bietti Foundation
  • , Graziana EspositoAffiliated withIRCCS-G.B. Bietti Foundation
  • , Alessandro LambiaseAffiliated withCIR, Department of Ophthalmology, University Campus Bio-Medico
  • , Stefano BoniniAffiliated withCIR, Department of Ophthalmology, University Campus Bio-Medico

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To assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery.


Human fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST–ANOVA coupled analysis was used to compare the molecular and biochemical data.


The fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis.


AM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.


Amniotic membrane Ocular surgery Toll-like receptors Growth factors Cytokines