Vibrational Microspectroscopy and Imaging of Molecular Composition and Structure During Human Corneocyte Maturation

Transcription

1 ORIGINAL ARTICLE Virtionl Microspectroscopy nd Imging of Moleculr Composition nd Structure During Humn Corneocyte Mturtion Guojin Zhng 1, Dvid J. Moore 2, Richrd Mendelsohn 1 nd Crol R. Flch 1 The outermost region of the epidermis, the strtum corneum (SC), provides n essentil rrier to wter loss nd protects ginst exogenous sustnces. The functionl integrity of the SC depends on complex mturtion nd exfolition process, which is often pertured in skin diseses. The mturtion of corneocytes isolted from different depths in helthy humn SC ws investigted using infrred (IR) spectroscopic imging nd Rmn microscopy. Both IR nd Rmn spectrl qulity of individul corneocytes ws high nd reveled depth-dependent vritions in moleculr composition. Spectrl chnges were identified s rising from ltertions in the concentrtion of the mjor constituents of nturl moisturizing fctor (NMF), importnt in mintining SC hydrtion. A significnt decrese in the concentrtion of NMF ws oserved for corneocytes isolted from superficil compred to deeper SC lyers (lyer 3 vs. lyer 11, respectively). An IR prmeter tht mesures the reltive NMF concentrtion in corneocytes is introduced. The potentil role of virtionl imging to evlute corneocyte composition nd moleculr structure in the tretment of NMF-relted diseses is discussed. Journl of Investigtive Dermtology (2006) 126, doi:.38/sj.jid ; pulished online 2 Mrch 2006 INTRODUCTION The superficil region of the epidermis, the strtum corneum (SC), provides vitl physicl rrier tht protects ginst externl insult nd mintins wter homeostsis. Indeed, primry function of the epidermis is to generte the SC. The sic structure of this thin (B 20 mm) outer lyer consists of kertin-rich corneocytes emedded in highly ordered lipid lmellr phse, nlogous in mcroscopic rchitecture to rick wll (Elis, 1991; Schefer nd Redelmeier, 1996). The hydrtion level in the SC, lthough low compred to the underlying vile epidermis nd dermis, is key to proper skin rrier function. Wter ctivity is thought to influence the mturtion of the SC vi its impct on enzyme ctivity nd lipid phse ehvior. Continul renewl of the SC is lso essentil for its functionl integrity. Corneocytes re generted from terminlly differentited kertinocytes s the cells mke their wy from the strtum grnulosum into the SC. During this trnsition, profilggrin, lrge, insolule protein, is converted to filggrin, which is 1 Deprtment of Chemistry, Newrk College of Arts nd Sciences, Rutgers University, Newrk, New Jersey, USA nd 2 Interntionl Specilty Products, Wyne, New Jersey, USA Correspondence: Dr Crol R. Flch, Deprtment of Chemistry, Newrk College of Arts nd Sciences, Rutgers University, 73 Wrren Street, Newrk, New Jersey 072, USA. E-mil: flch@ndromed.rutgers.edu Arevitions: IR, infrred; NMF, nturl moisturizing fctor; SC, strtum corneum Received 20 Septemer 2005; revised 21 Decemer 2005; ccepted Jnury 2006; pulished online 2 Mrch 2006 ssocited with the corneocyte kertin filments in the deepest lyers of the SC. Further processing of filggrin ppers to e driven y the wter grdient within the SC nd in helthy skin, the reltively low hydrtion level promotes proteolysis of the protein into its constituent mino cids, mino-cid derivtives, nd slts (Scott nd Hrding, 1986). The resulting mixture, known s nturl moisturizing fctor or NMF, is highly wter solule nd cn e s much s % of corneocyte dry weight. Atmospheric wter is sored y the hygroscopic NMF providing sufficient hydrtion to help keep skin flexile nd to fcilitte vrious enzymtic rections in the pthwy tht termintes with corneocyte desqumtion (Rwlings et l., 1994). The moleculr nture of this complex process in helthy nd disesed skin continues to e focus of skin reserch. The forementioned processes hve een investigted with cell culture nd immunohistochemicl techniques. In ddition, vrious opticl microscopic methods hve een developed to chrcterize the morphology of the SC nd underlying epidermis in helthy nd disesed skin. Techniques such s confocl lser scnning microscopy (Lngley et l., 2001; Gerger et l., 2005) nd two-photon fluorescence microscopy (So et l., 2000; Hnson et l., 2002; Yu et l., 2003) cn e pplied in noninvsive mnner, voiding the need to microtome thin sections while chieving high sptil resolution (to the sumicron level). Opticl microscopic techniques hve the potentil to ecome routine dignostic tools, lthough moleculr level informtion is limited nd exogenous proes my pertur ntive skin. 88 Journl of Investigtive Dermtology (2006), Volume 126 & 2006 The Society for Investigtive Dermtology

2 Virtionl spectroscopic imging nd microscopy offer significnt dvntges in this regrd. Proe molecules re unnecessry nd the techniques re sensitive to moleculr composition, structure, nd interctions mong moleculr components. However, sptil resolution is lower thn in fluorescence-sed methods (i.e., B mm in the infrred (IR) nd 1 3 mm in the Rmn) nd the depth of smpling is limited s well. Confocl Rmn microscopic mesurements cn e performed on intct skin to depths of B0 150 mm (Cspers et l., 2003; Xio et l., 2004, 2005). In generl, microtomed smples (B5 mm thick) re required for IR imging. Recent dvnces in IR spectroscopic imging nd confocl Rmn microspectroscopy hve permitted the cquisition of sptilly resolved chemicl composition nd structurl informtion from iologiclly importnt smples (for reviews, see Slzer et l. (2000) nd Vir Spectrosc 2005; 38) The vriety of iologicl smples nd methods hve expnded from tissues nd fixed cells to living cells (Moss et l., 2005). Chllenges in dt collection nd interprettion re eing confronted nd surmounted s the work moves from serving s n djunct to histopthology to plying more predictive role in dignosing disese, providing insights into the moleculr interctions preceding the onset of disese, nd trcking the effectiveness of therpeutics on moleculr level. In the current study, the mturtion process of individul humn corneocytes otined from different depths in the SC ws investigted using IR spectroscopic imging nd Rmn microspectroscopy. The results from experiments conducted on cells isolted from helthy skin revel depth-dependent compositionl differences nd provide n initil seline from which n exmintion of the vriility mong sujects, ody sites, ge, nd vriety of disese sttes cn e mde. RESULTS A visul microgrph of severl unstined corneocytes isolted from the tpe strip is presented in Figure 1. The corneocytes disply chrcteristic polygonl shpes nd sizes. An imge of the IR Amide I nd (B1650 cm 1 ) intensity for single corneocyte from the sme lyer is shown in Figure 1, the outer edges of which lso disply typicl corneocyte size. In the imge, rightness corresponds to more intense Amide I nd. The smll squre (solid line) within the IR imge denotes the pixel ( mm) from which the spectrum shown to the right (leled ) ws cquired. A representtive IR spectrum from the tpe strip is lso presented in Figure 1c. The spectrl fetures provide informtion out the composition of the cells nd the moleculr structure of prticulr constituents. The spectr demonstrte high signl-to-noise rtios. Only liner seline correction ws pplied to the spectr efore sttisticl nd dditionl nlysis. Severl strong nds chrcteristic of proteins nd lipids re oserved in the spectr including, N H stretching or Amide A (B3280 cm 1 ), CH 2 nd CH 3 stretching ( cm 1 ), Amide I (predominntly peptide ond C ¼ O stretch t B1650 cm 1 ), nd Amide II (B60% N H end nd 40% C N stretch t B1550 cm 1 ) Micrometers Micrometers c Asornce 0.05 nds. In ddition, firly strong nd is oserved t B1400 cm 1 only in the spectrum of the corneocyte otined from the tpe strip. The origin of this nd nd other differences etween corneocytes isolted from two depths within the SC will now e more closely exmined. IR imges were cquired from multiple corneocytes isolted from the nd tpe strips. The imges were sptilly msked y selecting 4 4 pixel re from the center of ech corneocyte s depicted y the dshed ox drwn in Figure 1. In this wy, intensity vritions owing to edge effects re minimized. A men spectrum ws clculted from the msked imges cquired of 36 corneocytes isolted from ech lyer. Men spectr ( cm 1 ) long with difference spectrum (lyer 11 lyer 3) re shown in the top of Figure 2. Before sutrction, the men spectr were normlized with respect to the intensity of the nd oserved t B1245 cm 1. This procedure lso effectively normlized the spectr with respect to oth the Amide I nd II modes (shown efore normliztion in Figure 2). Thus, the nd t 1245 cm 1 is resonly ssigned to the peptide ond Amide III mode (Krimm nd Bndekr, 1986). In Figure 2, significnt differences re oserved etween the two men spectr, the most prominent eing the incresed intensity in the nd t 1404 cm 1 in the spectrum cquired from corneocytes isolted from the deeper lyer. The croxylte symmetric stretching mode sors in this spectrl region. As the rekdown products of filggrin (NMF), for exmple, mino-cid slts nd ionized croxylic cid derivtives, re rich in this functionl group, spectrum ws cquired of dried film consisting of mjor NMF components (Figure 2, ottom spectrum). The fetures oserved in the difference Wvenumer/cm 1 Figure 1. Opticl nd IR imging of humn corneocytes. () Opticl imge of severl unstined corneocytes isolted from the tpe strip. Br ¼ 20 mm. () IR imge of Amide I nd intensity t 1650 cm 1 for single corneocyte from n lyer tpe strip. The righter res in the imge correspond to higher Amide I nd intensities. The lrge ox (dshed line) drwn in the center of the imge mrks the 4 4 pixel re (25 25 mm) from which corneocyte spectr were nlyzed. The smll ox (solid line) mrks the single pixel from which the corresponding spectrum shown in (c) ws otined. (c) Representtive IR spectr ( cm 1 nd cm 1 region) of corneocytes isolted from the (ottom spectrum) nd (top spectrum) tpe strips. Ech spectrum ws otined from single mm pixel. 89

3 Asornce Difference NMF film Wvenumer/cm 1 Wvenumer/cm 1 Figure 2. Men IR imging spectr of multiple corneocytes from two depths in humn SC compred to IR spectrum of model NMF film. () Men IR spectr ( cm 1 region) of 36 corneocytes isolted from lyer nd lyer tpe strips (top two spectr) long with the difference spectrum ( ) nd spectrum of model NMF film. () Men IR spectr from the sme two lyers in the Amide I nd II region. The rrow mrks the pproximte position of the symmetric croxylte stretching mode. spectrum re quite comprle to those in the spectrum of the dried film, thus corroorting ssignment of the nd t 1404 cm 1 to the croxylte symmetric stretch of NMF constituents. Qulittively, the concentrtion of NMF in corneocytes ppers to increse significntly with SC depth for the outermost B11 lyers. Men corneocyte spectr displyed over the cm 1 region in Figure 2 lso support NMF depth-dependent concentrtion differences s evidenced y incresed intensity in the corresponding croxylte symmetric stretching region (B1590 cm 1 ) oserved for corneocytes from the deeper SC lyer. This ppers to e the mjor difference etween the spectr shown in Figure 2 where the Amide I nd II nd shpe nd position re very similr for the cells from the two SC lyers. The similrity etween the two men spectr indictes tht the secondry structure of kertin, y fr the mjor contriutor to sornce in the Amide I nd II region, is essentilly the sme in the corneocytes isolted from the two depths. Helicl secondry structure, s is predominntly found in epiderml kertin, is indicted y the frequency of the Amide I nd centered t 1650 cm 1 in oth men spectr. It should e noted tht frction of the intensity in this region might lso e due to the Amide I nd II virtionl modes from cermide polr regions. A rnge of frequencies hs een reported for cermide Amide I modes (B cm 1 ) depending on the prticulr cermide nd/or model system studied (Moore et l., 1997; Rerek et l., 2005). In isolted corneocytes, however, sornce in this region owing to cermides is expected to e reltively low compred to kertin Amide I sornce. Msked IR imges from 72 corneocytes, 36 from ech lyer, were conctented to produce the imges shown in Figure 3. Within ech imge, the top two rows re from the corneocytes isolted from the tpe strip nd the ottom two rows from the tpe strip. The imges re correltion coefficient mps clculted over the cm 1 region etween ech men spectrum (shown in Figure 2) nd the Correltion to men spectrum Correltion to men spectrum Figure 3. Correltion coefficient imges of multiple corneocytes from two depths in humn SC. Sptilly msked (4 4 pixel re) conctented IR imges of correltion coefficients clculted for 72 corneocytes. In ech imge, the top two rows (totl of 8 pixels verticlly) contin correltion coefficients for corneocytes isolted from lyer tpe strips nd the ottom two rows re for corneocytes from lyer tpe strips. () Correltion to the men spectrum ( cm 1 region) from the lyer. () Correltion to the men spectrum ( cm 1 region) from the lyer. The color r shows the rnge of clculted correltion coefficients. spectrum from ech pixel in the originl full dt set. The correltion coefficients rnge from B0.7 (drk lue) to 1.0 (drk red). Correltion to the men generted from the lyer (Figure 3) nd similrly for the men from the lyer (Figure 3) clerly differentites the lyer from which the mjority of the corneocytes were isolted. Some inhomogeneity etween the cells isolted from ech lyer is evident in the imges. In prticulr, two outliers re pprent in the spectr of the corneocytes isolted from the tpe strip (Figure 3, top two rows, left side of imge). The vriility is thought to reflect the rnge of composition nd structure found in ntive corneocytes nd lso to result from the ccrul of rtifcts from sequentil tpe stripping. Finlly, reltively smll degree of vriility is oserved within mny of the individul corneocytes (ech consisting of 4 4 pixel re within the imges). To investigte the potentil effects of hexne wshing on corneocytes, different protocol ws used to isolte nd exmine five corneocytes from two SC depths. Using the procedure referred to s the direct method (see Mterils nd Methods), IR imges were cquired of the sme corneocyte efore nd fter hexne dousing. Representtive men spectr from single corneocytes (4 4 pixel re) otined fter tpe stripping two (superficil) nd (deeper) times re displyed in Figure 4 efore nd fter wshing with hexne. Evidently, hexne wshing does not produce significnt chnges in the spectrl region ( cm 1 ) shown in Figure 4. Some differences re oserved, however, in spectrl regions where lipids hve strong sorption nds (e.g., Figure 4, cm 1 ). For ech corneocyte, Amide I nd intensity ws essentilly unchnged (o1%) y the hexne tretment (not shown); however, some vrition Journl of Investigtive Dermtology (2006), Volume 126

4 Asornce Deeper Hexne + Hexne Deeper Rmn intensity (photon counts/second) 500 Superficil Superficil Wvenumer/cm 1 Wvenumer/cm 1 Figure 4. Men IR spectr of corneocytes from two depths in humn SC isolted using the direct method efore nd fter wshing with hexne. Men IR spectr from 4 4 pixel re of the sme corneocyte otined fter tpe stripping two (superficil) nd (deeper) times efore nd fter hexne tretment. () cm 1 spectrl region nd () cm 1 spectrl region. etween corneocytes within ech lyer ws oserved (B%). In ddition, Amide I nd II nd positions nd shpes did not chnge, indicting tht the secondry structure of kertin is unltered y hexne. The nd re rtio of the 1404 cm 1 croxylte mode to the Amide I virtion ws clculted nd verged for the five cells isolted from ech lyer providing semiquntittive nlysis of reltive NMF concentrtion. After hexne tretment, the reltive NMF concentrtion decresed y B1.5% for corneocytes from the superficil lyer nd B3.0% for the deeper lyer. When compred to the differences in the reltive NMF concentrtion etween the two lyers (40%) for cells isolted using oth methods, the decrese owing to hexne is insignificnt. Upon exmintion of the CH stretching region (Figure 4), lipid loss of 25 30% is detected in the spectr of corneocytes from oth lyers s determined from the nd re rtio of the symmetric methylene stretching mode (B2848 cm 1 ) to the Amide I. It is cler from the figure tht the symmetric methylene stretching mode (B2920 cm 1 ) ehves similrly. Evidently, this hexne-induced prtil loss of noncovlently ound lipid does not promote significnt leching of the NMF components. In ddition, the frequencies of oth methylene stretching modes indictive of cyl chin conformtionl order do not chnge significntly (o0.5 cm 1 ) fter hexne wshing. Overll, hexne ws found to ffect the corneocytes from oth lyers in similr mnner. Further confirmtion of the depth-dependent NMF loss ws provided y Rmn microspectroscopic mesurements of severl corneocytes isolted from the nd tpe strips. Men Rmn spectr for B8 corneocytes isolted from ech of two SC lyers ( nd tpe strips) re presented in Figure 5. In the figure, the sterisks mrk four spectrl regions (882, 1342, , nd 1650 cm 1 ) where incresed nd intensities re oserved for corneocytes isolted from deeper SC lyers. Previous reports from the Puppels l sed on Rmn mesurements of predominnt NMF components hve ssigned the feture t B1415 cm 1 to the mino cids, serine, glycine, nd lnine, nd the feture t B885 cm 1 to pyrrolidone-5-croxylic cid (Cspers et l., 1998, 2002), metolic product derived Rmn shift (cm 1 ) Figure 5. Men Rmn spectr of humn corneocytes from two SC lyers. Men Rmn spectr ( cm 1 region) of corneocytes isolted from (ottom spectrum) nd (top spectrum) tpe strips otined from humn forerm skin. The sterisks mrk nds or regions ssigned to NMF where reltive intensity significntly increses with SC depth. from the glutmte nd/or glutmine residues found in filggrin. The nd t B1650 cm 1 rises from vrious skin constituents nd t lest two overlpped virtionl modes. Contriutions to the Amide I mode re mde y proteins, cermides, nd specific NMF constituents such s, pyrrolidone croxylic cid, which contins n mide group. The C ¼ C stretching mode lso produces nd in this region, reltively shrper nd stronger thn the Amide I nd. A mjor NMF component, urocnic cid, derived from the mino cid histidine, contins C C doule ond conjugted with the histidine imidzole ring. The mino-cid composition of filggrin includes pproximtely 5% histidine residues (Scott et l., 1982). The significnt difference oserved in the Rmn intensity of the B1650 cm 1 nd my in prt e due to depth-dependent chnges in protein, cermide, nd pyrrolidone croxylic cid concentrtions; however, lrger portion of the vrition is suggested to rise from decrese in the mount of urocnic cid present in the most superficil SC lyers. The reltively smller depth-dependent chnge oserved in the IR Amide I nd intensity (Figure 2) supports this interprettion given tht the C ¼ C stretching virtion is very wek in the IR. In relted reports from the Puppels group, in vivo confocl Rmn mesurements provided reltive concentrtion depth profiles for totl NMF nd severl of its individul components in humn skin from two different ody sites oth with reltively thick SC (B70 0 mm) (Cspers et l., 2001, 2003). Concurrent with the in vivo mesurements, the increse in the intensity of the nds with depth oserved in the current Rmn nd IR experiments lmost certinly reflects n increse in corneocyte NMF concentrtion in going from superficil SC regions ( tpe strip) to the B lyer of cells. An NMF concentrtion profile ws constructed from IR imges of individul corneocytes isolted from the, 6th, nd tpe strips using the rtio of integrted nd res, 1404 cm 1 /Amide I. IR imges of the nd re rtio for the three corneocytes re shown in Figure 6. A plot of this spectrl prmeter cquired long the white line drwn in Figure 6 long with the men vlue for ech corneocyte re displyed in Figure 6. The dt from the three corneocytes 91

5 c Reltive NMF concentrtion th show tht the reltive concentrtion of NMF more thn doules with depth in the outermost pproximtely 11 lyers of the SC (from 0.41 to 0.88). These results re in generl greement with the outer lyers smpled in the confocl Rmn study mentioned ove (Cspers et l., 2003) nd in excellent greement with n erlier review depicting the concentrtion of one of the mjor components of NMF, pyrollidone croxylic cid, in the outer 8 mm of the SC (Rwlings et l., 1994). An imge of the sme nd re rtio, clculted for the full corneocyte dt set used herein, is presented in Figure 6c with the rnge of vlues shown in the color r. The men rtio for the lyer is with stndrd devition of nd for the lyer. The IR spectrl prmeter provides mesure of reltive NMF concentrtion tht is oserved to differ significntly with SC depth. DISCUSSION Significnt efforts re underwy to develop virtionl spectroscopic imging pplictions tht re useful to the Figure 6. Reltive NMF concentrtion profile determined from IR imging of corneocytes isolted from different depths in humn SC. () Reltive NMF concentrtion imge determined from the rtio of integrted nd res (1404 cm 1 /Amide I) in three corneocytes isolted from the, 6th, nd tpe strips. The color red corresponds to higher concentrtion nd lue to lower. () Reltive NMF concentrtion s determined y the integrted nd re rtio (1404 cm 1 /Amide I) long the white line drwn in (). The individul dt points represent the mesured rtio otined from ech pixel in the imge nd the solid lines re the men vlues for ech corneocyte. (c) Imge of reltive NMF concentrtion for the full corneocyte dt set s descried in the cption for Figure 3. iomedicl community. Severl pproches re eing tken to investigte the competency of the techniques for detecting the initil stges of disese, understnding moleculr chnges ccompnying disese, nd defining the effects of therpeutic intervention on moleculr level. Towrds this end, the ility to delinete iochemicl composition nd moleculr events within single cell should prove very useful. Acquiring moleculr level informtion using in vivo smpling is nother vlule pproch. The current work demonstrtes the fesiility of cquiring moleculr level iochemicl nd structurl informtion from single corneocytes smpled t different points in the mturtion nd exfolition process. In helthy skin, the complex process of complete cell turnover tkes B30 dys. Most notly, the present work revels the depth dependence of NMF concentrtion in the outer B6 mm of the SC. NMF components re thought to fcilitte criticl iochemicl events in the SC. Perturtions in NMF concentrtion hve een linked to ging, surfctnt exposure, nd disese sttes of vrying severity. Thus, the quntifiction of the NMF concentrtion grdient in the SC will provide meningful informtion to the medicl, phrmceuticl, nd cosmetic industries. In vivo confocl Rmn mesurements of humn skin hve een reported delineting NMF nd wter concentrtion profiles t ody sites known to hve thick SC (Cspers et l., 2002, 2003). This noninvsive pproch coupled with confocl lser scnning microscopy hs shown tht oth profiles re directly relted to skin rchitecture (Cspers et l., 2003). The results re consistent with n erlier report finding tht decrese in wter concentrtion is required efore filggrin proteolysis to form NMF (Scott nd Hrding, 1986) s occurs t the junction of the SC nd vile epidermis. The in vivo Rmn mesurements were conducted using depth increments of mm up to totl depth of B150 mm from the skin surfce. The methodology used in the current work essentilly increses the xil resolution (in the superficil SC region smpled), s one tpe strip removes pproximtely one lyer of corneocytes (B0.5 mm). In generl, the consistency etween the results of the different smpling protocols nd spectroscopic methods provides high level of confidence in the smpling nd mesurement protocols used in oth lortories. Skin pthologies such s psorisis, topic dermtitis, nd ichthyosis vulgris disply norml desqumtion processes where the mount of NMF is gretly decresed or is sent (Rwlings et l., 1994). In report compring SC iopsies from the heels of helthy sujects nd from the symptom-free heels of ptients with ctive psorisis, Rmn spectr show significnt decrese in the reltive intensity of nd t B1418 cm 1 (Wohlr et l., 2001), which my e interpreted s decrese in NMF. A more recent Rmn study compring helthy nd psoritic skin reported disruption in lipid conformtionl order nd suggested unfolding of proteins in psoritic smples (Osd et l., 2004). In psorisis, it is not entirely cler whether mlfunctioning rrier is the driving force or the consequence of kertinocyte hyperprolifertion nd immune-medited inflmmtory responses (Ghdilly et l., 1996; Lewohl, 2003). In either cse, 92 Journl of Investigtive Dermtology (2006), Volume 126

6 spectrl prmeters such s tht derived to mesure reltive NMF concentrtions, long with estlished mrkers of protein secondry structure nd lipid order could e used to evlute therpeutic interventions. Virtionl imging techniques my not only prove useful in evluting disese sttes, ut s demonstrted here, my lso clrify results otined using common procedures. Orgnic solvents nd other gents re often used s permetion enhncers or in experimentl protocols to evlute rrier function. The effects of prticulr gents on extrcting SC lipids hve een reported (Arms et l., 1993), ut it is generlly more difficult to ccess moleculr structure ltertions. In the current work, use of the solvent hexne is shown to preserve corneocyte NMF concentrtion nd kertin secondry structure while prtil removl of noncovlently ound lipid occurs (Figure 4). Although the hexne-induced extrction of some SC lipids ws nticipted from previous reports on skin iopsies (Arms et l., 1993), the mintennce of kertin structure nd NMF concentrtion ws unknown. These initil studies identify severl IR nd Rmn spectrl prmeters to evlute reltive NMF nd lipid concentrtions together with lipid nd protein conformtion in individul corneocytes. Additionl smpling with vritions in gender nd ge of sujects, for instnce, will id in ssessing the sttisticl roustness of the prmeters. Further investigtions my lso provide clerer picture of the corneocyte mturtion process from which vrious therpeutic interventions in the tretment of skin disorders cn e evluted. MATERIALS AND METHODS Preprtion of corneocytes The Rutgers University Internl Review Bord hs pproved ll protocols used herein. The ethicl principles for nonclinicl iomedicl reserch involving humn sujects s declred in the Declrtion of Helsinki Principles were dhered to nd consent from the suject ws otined. Corneocytes from different lyers of the SC were collected from the sme site of humn forerm skin y sequentil tpe stripping (Sellotpe, 3 M Scotchgurd) n re of pproximtely cm. The forerm re ws flushed with wter for severl minutes efore tpe stripping. The first two tpe strips were discrded nd corneocytes collected from the, 6th, nd tpe strips were prepred s follows. Corneocytes were flushed off the tpe into eker using HPLC grde hexne. The corneocyte/ hexne suspension ws sonicted for 5 minutes to rek up the desmosoml nd lipid cohesion etween individul corneocytes. The corneocytes were then isolted using nitrocellulose memrne filters (GE Osmonics Lstore, Minnetonk, MN; pore size 0.22 mm). Corneocytes were rinsed nd resuspended using fresh hexne. This procedure ws repeted three times to ensure tht ll glue hd een removed. Corneocytes were hrvested nd isolted on severl seprte occsions. Aliquots of this corneocyte suspension, contining some noncovlently ound lipids, were deposited on either CF 2 windows for IR imging or gold-coted silicon sustrtes for Rmn mesurements. The mjority of the results presented herein used this method for corneocyte isoltion. All smples were dried overnight under house vcuum efore mesurements were mde. A smller numer of corneocytes were isolted using more difficult protocol, referred to s the direct method, to specificlly evlute the effect of hexne on individul corneocyte composition nd the moleculr structure of cell constituents. This method is inconvenient for scling up to evlute the numers of corneocytes necessry for sttisticl dt nlysis, ut it does serve s n pproprite protocol for the nlysis of control smples. Corneocytes were directly collected from humn forerm skin y pressing CF 2 window on the site fter tpe stripping nd gently ruing the skin with cotton tip. A few free corneocytes stuck to the window fter tpe stripping the site two nd times. Corneocytes re designted s eing isolted from either superficil or deeper SC lyer (see Results), s it seems s if the ssignment of corneocytes to specific SC lyer is less certin compred to the first tpe stripping protocol descried ove. Using the light microscope portion of the Perkin- Elmer Spotlight instrument, single corneocytes were mrked nd IR imges were cquired of the sme corneocyte efore nd fter continuously soking the cell with smll liquots of hexne (totl volume 50 ml) for pproximtely minutes. A finl ml hexne flush ws pplied to ech corneocyte efore drying the smple nd IR dt collection. Residul hexne ws not detected in the spectr. Preprtion of NMF film Chemicls were purchsed from Sigm-Aldrich (St Louis, MO) nd the dry powders were comined s follows (in weight %): serine (30%), glycine (18%), pyrrolidone croxylic cid (16%), lnine (11%), histidine (7%), ornithine (6%), citruline (5%), rginine (5%), nd proline (2%). These reltive concentrtions roughly reflect the mino-cid composition of filggrin (Rwlings et l., 1994) nd re in generl greement with previously pulished in vivo studies of humn NMF composition (Cspers et l., 2001). A solution ws prepred in distilled wter t ph 5.5, spred on to CF 2 window, nd llowed to dry overnight t room temperture to gel-like film. IR imging IR microscopic imges were cquired with the Perkin-Elmer Spotlight system, which consists of n essentilly liner rry (16 1 detector elements) mercury cdmium telluride detector long with n utomted high-precision XY smple stge. Aout 50 corneocytes from ech lyer were rndomly selected nd IR imges (B60 60 mm 2 ) were cquired with pixel size of 6.25 mm nd sptil resolution of 12 mm. Thirty-two scns were collected for ech spectrum using 8 cm 1 spectrl resolution nd one level of zero-filling yielding dt encoded t 4 cm 1 intervls. Ech corneocyte imge ws cquired in B20 minutes. Rmn microscopy Rmn spectr of corneocytes were cquired with Kiser Opticl Systems Rmn Microproe. The instrument hs confocl smpling cpility nd hs een previously descried in detil (Xio et l., 2004). A solid-stte diode lser (785 nm excittion wvelength) genertes B4 7 mw of single mode power t the smple. The ckscttered light is collected from spot size of B2 mm nd illumintes thermoelectriclly cooled, ner-ir CCD (ANDOR Technology, Model DU 401-BR-DD). Spectrl coverge is from 0 to 3450 cm 1 t spectrl resolution of 4 cm 1 nd dt re encoded every 0.3 cm 1. Acquisition time for ech spectrum ws B minutes (including 93

7 cosmic-ry correction) nd 15 corneocytes from ech lyer were smpled. Dt nlysis Grms/32 AI softwre version 6.0 (Thermo Glctic, Slem, NH) ws used for processing Rmn spectr. Rmn spectr from ech lyer were verged, Fourier smoothed (80%), nd liner selines were pplied. Processed men spectr were compred to the rw dt to ensure tht spectrl fetures were not misrepresented. As the originl dt re encoded every 0.3 cm 1, the reltively lrge degree of Fourier smoothing could e pplied without distorting the spectr. Spectrl Dimensions (Olney, MD) ISys 3.0 softwre ws used for the nlysis nd construction of the IR imges. Liner selines were pplied over spectrl regions of interest. Dt interprettion ws fcilitted y verging the spectr of corneocytes from prticulr SC lyers to improve signl-to-noise rtios. Correltion coefficients were then clculted etween the men spectrum of ech lyer nd ech spectrum contined in n imge to generte correltion mps. CONFLICT OF INTEREST The uthors stte no conflict of interest. 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