Bina Kulkarni

Morphological characteristics of the limbal epithelial crypt

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells. Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus. Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3−/CK19+/CD 34−/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)−. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes. Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Limbal epithelial crypt: a model for corneal epithelial maintenance and novel limbal regional variations.

OBJECTIVE To determine the distribution of cell membrane proteins and extracellular matrix proteins around the limbal epithelial crypt (LEC) compared with adjacent limbus and corneal epithelium. METHODS Serial histological sections of human corneoscleral limbus rims were stained with antibodies of interest by standard immunohistochemistry. RESULTS Superficial cells of the limbus were desmoglein 3 positive, compared with the negative basal cells of the limbus that correspond to cells with more stemlike properties. The LEC had a much lower proportion of desmoglein 3 staining in comparison. Tenascin C staining demonstrated regional variations of the limbus depending on their association with the LEC. Limbus that was associated with or adjacent to the LEC had a greater tenascin C expression compared with normal limbus, whereas the LEC demonstrated the greatest tenascin C expression. CONCLUSIONS Based on these and similar results previously reported for connexin 43, we propose a novel model on the mechanism of corneal surface epithelium maintenance involving 3 different limbal regions: zone 1, limbus including the LEC; zone 2, limbus associated with the LEC; and zone 3, limbus distant to the LEC. CLINICAL RELEVANCE The noted limbal variations may influence the selection of the donor site for limbal grafts in the future.

Localization and Gene Expression of Human β-Defensin 9 at the Human Ocular Surface Epithelium

PURPOSE Antimicrobial peptides (AMPs) are multifunctional host defense molecules. Human beta-defensin 9 (HBD9) has previously been shown to be downregulated during ocular surface (OS) infection or inflammation. Here, the authors aimed to study localization of HBD9 protein in different OS regions and to determine the role of Toll-like receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors, and proinflammatory cytokines in HBD9 expression. METHODS Immunolocalization of HBD9 protein was carried out on the normal human OS regions (cornea, limbus, and conjunctiva). Quantitative PCR analysis of HBD9 mRNA was performed in SV40-transformed human corneal epithelial cells (hCECs) treated for different durations with synthetic pathogen-associated molecular patterns (PAMPs) and recombinant cytokines. RESULTS HBD9 protein was constitutively expressed on OS epithelia. Corneal and limbal epithelia and corneal stroma demonstrated modest levels of HBD9, whereas conjunctival epithelium demonstrated high levels of HBD9 protein. TLR02, TLR03, TLR04, and TLR05 were shown to modulate HBD9 mRNA in hCECs. Similarly, NOD2 and IL-1beta were also shown to alter HBD9 in a time-dependent manner. In response to infection-related PAMPs and inflammatory cytokines, an initial increase in HBD9 mRNA levels was observed, followed by a significant downregulation. CONCLUSIONS This is the first demonstration of HBD9 protein expression at different OS regions. The authors also determined the role of various innate immune receptors in HBD9 mRNA modulation. Further understanding of the signaling mechanisms involved in the initial response of HBD9 to infection or inflammation is likely to indicate future therapeutic directions with this AMP.

Validation of Endogenous Control Genes for Gene Expression Studies on Human Ocular Surface Epithelium

Purpose To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. Method The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG—glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)—was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. Results The expression stability of ECGs in the OS epithelial regions in increasing order as determined with geNorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. Conclusion This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium.

Quest for limbal stem cells.

pleted suggesting that the circumferentially migrating population of cells probably represented, in part, the healing response of limbal progenitor cells. More direct evidence of the significance of the limbus as a repository of progenitor or stem cells comes from observations on clinical pathology. In patients with limbal abnormalities not related to trauma, alternating columns of normal and fluorescein staining cells have been noted to extend from the limbus towards the centre in radial or curvilinear rows. The columnar arrangement (columnar keratopathy) can, in some patients, be seen to correspond with the limbal palisades. 5 A similar ‘streaming’ of fluorescein-stained cells in columns has also been observed in relation to broken sutures following corneal grafting. 5 These observations lend support to the belief that progenitor cell activity does not occur contiguously along the limbus but rather in an interrupted manner presumably corresponding to repositories of stem cells in the rete ridges that alternate with palisades. Disease or destruction of the corneoscleral limbus is associated with consequential events that can eventually lead to visual impairment or blindness. Collectively this is referred to as stem cell deficiency, or clinically more appropriately, limbal deficiency. A number of congenital and acquired conditions can cause limbal deficiency. 1,6 Limbal (stem cell) deficiency leads to alterations and aberrations in the corneal surface epithelium with a multitude of knock-on effects. The hallmark of limbal (stem cell) deficiency is ‘conjunctivalization’ of the cornea and the most significant clinical manifestation is a persistent corneal epithelial defect. 6

Corneal epithelial defects related to high postoperative astigmatism.

High and sometimes irregular astigmatism is not an infrequent complication following anterior segment surgery such as suturing of large wounds and corneal transplants. Abnormal corneal topography may affect tear film stability adversely. We present the case of two patients who had persistent postoperative epithelial defects in the lower third of the cornea, in the presence of high corneal astigmatism despite aggressive lubrication. Upon addressing their high corneal astigmatism their epithelial defects resolved. An 84 year old man underwent a repeat, HLA matched, penetrating keratoplasty following a failed graft for pseudophakic bullous keratopathy. He had had dry eye symptoms with his primary …