Disruption of bladder epithelium barrier function afterspinal cord injury - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：GerardApodaca, SusannaKiss, WilyRuiz, SusanMeyers, MarkZeidel,  LoriBirder,作者单位：1 Laboratory of Epithelial Cell Biology, Department of CellBiology and Physiology, Renal-Electrolyte Division,Department of Medicine, and Department of Pharmacology,University of Pittsburgh, Pittsburgh, Pennsylvania 15261 6 G, a8 I9 h# w. u( f3 p; g
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      【摘要】
   Neural-epithelial interactions arehypothesized to play an important role in bladder function. Wedetermined whether spinal cord injury (SCI) altered several indicatorsof urinary bladder epithelium barrier function, including continuity ofthe surface umbrella cell layer, transepithelial resistance (TER), andurea and water permeability. Within 2 h of SCI, significantchanges in uroepithelium were noted, including disruption of thesurface umbrella cells and an ~50% decrease in TER. By 24 h,TER reached a minimum and was accompanied by significant increases inwater and urea permeability. Regeneration of the surface uroepithelium was accomplished by 14 days after SCI and was accompanied by a returnto normal TER and urea and water permeabilities. This early disruptionof the uroepithelial permeability and accompanying changes inuroepithelial morphology were prevented by pretreatment withhexamethonium (a blocker of ganglion transmission), indicating involvement of sympathetic or parasympathetic input to the urinary bladder. In addition, prior treatment with capsaicin worsened theeffect of SCI on uroepithelial permeability, suggesting that capsaicin-sensitive afferents may play a protective role in the process. These results demonstrate that SCI results in a significant disruption of the urinary bladder uroepithelium and that these changesmay be mediated in part by an interaction with bladder nerves.
      【关键词】 uroepithelium urea and water permeability bladder nerves) E8 r5 H8 r* s. ~/ n  X
   INTRODUCTION
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THE STORAGE FUNCTION of the bladder depends on the presence of a barrier thatprevents urine from being reabsorbed into the bloodstream and regulatesthe expulsion of urine from the body ( 14, 16, 17, 23, 34 ).During bladder filling, the outlet, which includes the bladder neck andthe smooth and striated muscles of the urethra, remains closed and thesmooth muscle of the bladder remains relaxed ( 14, 16, 17 ).The uroepithelium, which lines the mucosal surface of the bladder,forms the permeability barrier, which retains the urine and preventsthe paracellular leakage of solutes and ions across the outermost,umbrella cell layer. Characteristics of the barrier include a hightransepithelial resistance (TER) and low permeability to urea and water( 27, 34 ). Intravesical pressure remains low duringfilling, and increased urine volume can be accommodated by unfolding ofthe mucosa and accompanying increases in the surface of the umbrellacell layer ( 51 ). When the bladder reaches capacity andvoiding is initiated, the muscles of the outlet relax, and urine isexpelled by contractions of the smooth detrusor muscle in the wall ofthe bladder.
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The interplay between the outlet and the bladder is under control ofthe central nervous system as well as sympathetic, parasympathetic, andsomatic nerves that innervate the detrusor muscle and outlet in areciprocal fashion ( 14, 16, 17 ). Bladder innervation canbe disrupted as a result of trauma or disease ( 14, 16, 17 ). For example, rostral lumbar spinal cord transectioneliminates voluntary control of voiding and is accompanied byuncoordinated bladder and external urethral sphincter activity, termeddetrusor-sphincter dyssynergia ( 7, 8, 10, 15, 16, 55 ). Anadditional consequence of spinal cord injury (SCI) is a highintravesical bladder pressure, which can lead to damage and malfunctionof the upper urinary tract and hypertrophy of the smooth muscle mass ( 56 ). The latter is accompanied by enormous enlargement ofthe urinary bladder ( 32, 56 ). Although a great deal ofattention has focused on alterations in the detrusor muscle and itsinnervation after SCI, much less is understood about changes in theuroepithelium and its associated barrier function that might accompany SCI." H' c( F# C! i( u( x0 L

An emerging body of literature indicates reciprocal communicationbetween the neuronal system and the uroepithelium. The urinary bladderis innervated by several types of bladder afferents (A - and Cfibers), some of which are found in the muscle layers and some justbelow and within the uroepithelium ( 5, 19, 33 ). Significantly, epithelium-associated afferent nerves are sensitive tochemicals released from uroepithelial cells, including nitric oxide(NO), prostaglandins, and ATP ( 2, 30, 36 ). The latter binds to P2X 3 purinergic receptors on afferent nerves andis likely to play an important role in detrusor contraction as well asnociception ( 40, 53 ). In turn, uroepithelial cells respondto various chemicals and neurotransmitters, including acetylcholine,norepinephrine, ATP, calcitonin gene-related peptide, and substance P( 3-6 ). Changes elicited in the uroepithelium by avariety of stimuli include the release of mediators such as NO and ATPand increases in intracellular Ca 2  ( 3, 5, 6, 18, 48, 53 ). Whether neurotransmitters affect parameters associated withuroepithelial barrier function, such as maintenance of a high TER orlow water and urea permeabilities, is not well understood, buttreatment with stress hormones, including norepinephrine, can leadto disruption of tight junctions and loss of umbrella cells( 52 ).4 c1 u. d: ]! y9 v; q/ s7 h

To examine the relation between the nervous system and theuroepithelium, we have studied the effects of SCI on uroepithelial cellmorphology as well as barrier function. We observed that SCI wasaccompanied by a rapid loss of barrier function, and these acutechanges were prevented by pretreatment with the ganglion transmissionblocker hexamethonium, whereas capsaicin potentiated the effects ofSCI. Normal barrier function was observed 14-28 days after SCI.Our results indicate that neurotransmitter release shortly after SCImay play an important role in disrupting epithelial barrier function.

MATERIALS AND METHODS
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Materials and animals. Unless specified otherwise, all chemicals were obtained from Sigma (St.Louis, MO) and were of reagent grade or better. [ 14 C]ureaand [ 3 H]water were obtained from American RadiolabeledChemicals (St. Louis, MO). Female Sprague-Dawley rats weighing275-350 g were fed a standard diet (Lab Diet 5P00, PMINutritional, Brentwood, MO) with free access to water before and afterSCI. All animal studies were carried out with the approval of theUniversity of Pittsburgh Animal Care and Use Committee, and animalswere maintained according to the standards set forth in the AmericanPhysiological Society's "Guide for the Care and Use of Animals."
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Rat model of SCI. Rats were anesthetized with halothane [2% (vol/vol) in oxygen], andafter an epidural injection of lidocaine (Xylocaine, 5 µl) alaminectomy was aseptically performed at theT 8 -T 9 spinal level. Gelfoam (Fisher) wasplaced between the cut ends of the spinal cord, and the muscle and skinwere sutured. The animals were allowed to recover in the absence ofanesthesia for 2 h, 1 day, 3 days, or 24 days. Sham-operatedanimals were placed under anesthesia, and the bone and musclesurrounding T 8 -T 9 were removed, but thespinal cord was not transected. Sham-operated and SCI rats were givenprophylactic antibiotic treatment (ampicillin, Polyflex, 100 mg/kg imdaily) for 1 wk after the procedure and allowed free access to food andwater. During the first 7-10 days after transection, urine waseliminated from the bladder by manual compression two to three times aday. After this time, spinal reflexes developed that allowed forautonomic control of micturition. To provide a control for effects ofbladder distension that might follow acute spinalization, somespinal-intact animals were placed under continuous halothane anesthesia(which suppresses bladder reflexes) for 2.5 h before euthanasiaand tissue removal. This amount of time allowed accumulation of urinevolumes that were similar to those observed 2 h after SCI. In oneanimal with SCI, urine volume was 3 ml. The data for this animal werediscarded, inasmuch as it was likely that the bladder wasoverdistended. In some experiments, animals were treated withhexamethonium (50 mg/kg iv; Sigma) 0.5 h before SCI. Other animalswere injected with 5 µl of PBS beneath the dura of the spinal cord(via a 30-gauge needle) into the underlying gray matter, and the needlewas removed without transection of the spinal cord. After incisionswere sutured, the animals were allowed to recover for 2 h in theabsence of anesthesia. In addition, a small number of animals( n = 4) were pretreated with capsaicin (100 mg/kg sc;Sigma) in 10% (vol/vol) ethanol-10% (vol/vol) Tween 80 in normalsaline 4 days before further experimentation. Desensitization wasconfirmed by measuring the number of defensive forelimb eye-wipemovements in response to 100 µg/ml capsaicin solution( 11 ). Control animals were given vehicle [10% (vol/vol)ethanol-10% (vol/vol) Tween 80 in normal saline] not containing capsaicin.+ T# u* Z0 L% W* b5 I+ S7 U+ T

Measurements of water permeability, urea permeability, and TER. Water permeability, urea permeability, and TER were measured asdescribed previously ( 24, 34 ). Briefly, after thetreatments described above and at the designated time points, theanimals were euthanized (by inhalation of medical-grade CO 2 followed by thoracotomy) and the bladder was placed immediately intoRinger solution (in mM: 111.2 NaCl, 25 NaHCO 3, 4.8 KCl, 2 CaCl 2, 1.2 MgSO 4, 1.2 KH 2 PO 4, and 11.1 glucose) maintained at 37°Cand constantly bubbled with 95% O 2 -5% CO 2 tomaintain pH at 7.4. The tissue was placed on a stretching rack with theepithelium facing downward. A ring with a 0.73-cm 2 openingand surrounded by sharp metal pins was placed under the tissue andlifted up to spear the overlying tissue. Excess tissue surrounding thering was removed with tissue scissors, and the ring and its associatedbladder tissue were mounted between two halves of a modified Ussingchamber. The chambers, filled with Ringer solution, were maintained at37°C, and the hemichambers were constantly stirred. The membraneswere allowed to stabilize for 1 h before electrical measurementsof TER to determine epithelial membrane integrity, and radiolabeledwater and urea were added to measure permeability. Significant changesin TER were assessed by t -test.

14 C-labeled (0.25 µCi/ml) urea and 3 H-labeled(1 µCi/ml) water were added to the apical (luminal) side of themembrane, and both hemichambers were sampled (2 × 100 µl perhemichamber) at 15-min intervals throughout the experiment.After 1 h of baseline measurements, the ionophore nystatin (185 µM) was added to the apical side to increase permeability across theapical membrane. To determine the contribution of unstirred layers tothe measured permeabilities, the apical membrane barrier was ablated byaddition of 100 µl of Triton X-100 1 h after addition ofnystatin. In all experiments, addition of nystatin and Triton X-100abolished TER. Fluxes were calculated as described previously( 24, 34 ). Significant changes in permeability wereassessed by t -test.

Scanning electron microscopy of uroepithelium. At the designated time points, rats were euthanized as described above,and the bladder was rapidly excised, cut along the dorsal margin, andplaced in a fixative containing 2.0% (vol/vol) glutaraldehyde and2.0% (wt/vol) paraformaldehyde in 100 mM sodium cacodylate, pH 7.4, 1 mM CaCl 2, and 0.5 mM MgCl 2 for 2-4 h at 4°C. The fixed tissue was washed for 15 min in 100 mM sodiumcacodylate buffer, pH 7.4, and then treated with 1% (wt/vol)OsO 4 in 100 mM sodium cacodylate buffer, pH 7.4, for 60 minat 4°C. After several rinses with distilled water, the tissue was cutinto small blocks (~0.5-mm cubes), and the samples were washed threetimes over 45 min in PBS and then dehydrated for 15 min in a graded series of ethanol: 30, 50, 70, and 95% (vol/vol in water). The sampleswere then incubated three times for 45 min in absolute ethanol. Thedehydrated samples were critical point dried, sputter coated withgold-palladium, and viewed in a Jeol JSM T300 scanning electronmicroscope at 20 kV. Images were captured on Kodak type 52 film,scanned on a Linotype-Hell Saphir Ultra II scanner, contrast correctedin Photoshop 6.0 (Adobe), assembled in Freehand 10.0 (Macromedia, SanFrancisco, CA), and output on a dye sublimation printer (model 8650PS,Kodak, Rochester, NY). All images are representative of similar resultsobtained from the bladders of at least three animals.
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Transmission electron microscopy of uroepithelium. Bladder tissue was fixed and osmicated as described above and then cutinto small blocks. After several water rinses, the samples were staineden bloc overnight with 0.5% uranyl acetate in water. Samples weredehydrated in a graded series of ethanol, embedded in epoxy resin(LX-112, Ladd), and sectioned with a diamond knife (Diatome). Sections,silver to pale gold in color, were mounted on butvar-coated coppergrids, contrasted with uranyl acetate and lead citrate, and viewed at80 kV in a Jeol 100 CX electron microscope. Images were printed andprocessed as described above. All images are representative of similarresults obtained from the bladders of at least three animals.8 V: o# _( H, A

RESULTS

SCI is accompanied by decreases in TER, water permeability, andurea permeability. The uroepithelium forms a barrier that is characterized by its highresistance to ion flow and low permeabilities to water and urea( 24, 28, 34 ). One indication of barrier function is TER,which measures the ability of the epithelium to impede paracellular(across the junctions) and transcellular (across the cell) ion flow. Inumbrella cells, the combined low levels of paracellular ion flow andmodest transcellular ion flow result in a high TER.

As controls, the TER was measured in animals that underwent no priorexperimental manipulation. Sham controls were placed under anesthesia,and bone around the spinal cord was removed, but the spinal cord wasnot transected; then the animals were allowed to recover for up to 28 days. Distension controls were allowed to fill their bladders for2.5 h but were unable to void because of anesthesia. The volume ofurine in animals 2 h after SCI was 1.63 ± 0.4 ml, and indistension controls the urine volume was 1.53 ± 0.05 ml after2.5 h. The TER values for all these controls were essentiallyidentical (i.e., 1,802 ± 101 · cm 2; Fig. 1 A ) and, as such, were groupedand are collectively referred to as "controls" in Figs. 1 and 4.This value is lower than those measured in rabbits but similar to ourprevious observations and may represent species variation, differencesin the methods of measurement, or differences in the diet of theanimals ( 24, 28, 34 ).4 `7 p$ U9 ^+ ~" O7 ~% l

Fig. 1. Effect of spinal cord injury (SCI) on transepithelialresistance (TER, A ), water permeability ( B ), andurea permeability ( C ). Control bladders or bladders fromanimals 2 h-28 days after SCI were mounted in Ussing chambers, andTER, water permeability, and urea permeability were monitored. Valuesare means ± SE from 4-32 bladders. * P

To assess the impact of SCI on TER, the spinal cord of anesthetizedrats was transected at T 8 -T 9. The animalswere allowed to recover for 2 h, 24 h, 3 days, 14 days, or 28 days, and then the bladder was excised and TER of the organ tissuemounted in Ussing chambers was measured. We observed a significantdecrease in TER as early as 2 h after SCI (965 ± 98 · cm 2, P above (Fig. 1 A ). Thelargest decrease in TER was observed at 24 h, when TER was466 ± 28 · cm 2. However, by 3 days afterSCI, TER was beginning to recover, and, by 28 days, TER approachedcontrol levels of 1,771 ± 590 · cm 2 (Fig. 1 A )., U/ }. R! y& ?6 S1 h

Next, we measured the impact of SCI on water and urea permeability.Controls identical to those used in the TER analysis were employed inthese studies. As we reported previously, water and urea permeabilitieswere low in control tissue: ~2.3 × 10 5 or2.2 × 10 6 cm/s, respectively ( 24, 34 ).Water permeability remained near control levels after 2 h butsignificantly increased ~2.5-fold above controls after 24 h(Fig. 1 B ). At 3 days, water permeability was increased3.7-fold above controls (Fig. 1 B ). Water permeability decreased to near control levels after 14 days and remained at theselevels up to 28 days after SCI (Fig. 1 B ). Urea permeability followed a similar pattern. At 2 h after SCI, there was little alteration in urea permeability; however, by 24 h a 2.7-foldincrease in urea permeability was observed (Fig. 1 B ). Ureapermeability was elevated at 24 h and after 3 days but was notsignificantly different from controls 14 or 28 days after SCI (Fig. 1 B ).4 U' `- M! b# ^! K3 B3 Z* m

SCI is accompanied by disruption of the uroepithelium. Scanning and transmission electron microscopy (SEM and TEM,respectively) were used to determine whether the functional changes inthe uroepithelium of SCI animals were accompanied by morphological changes in the uroepithelium of these animals. The ultrastructure ofumbrella cells from sham-operated animals (control) is shown in Figs. 2 A and 3 A. Identical morphology wasobserved in untreated animals and distension controls and is not shown.When examined by SEM, the umbrella cells of these control animals werelarge (~40-50 µm across) and polyhedral in shape, and thesurface membrane was folded in a parallel array (Fig. 2 A ). Athin tight junction complex circumscribed the perimeter of each cell(Fig. 2 B ). In thin-section electron micrographs, theuroepithelium contained an upper umbrella cell layer, two to threeintermediate cell layers, and a basal cell layer that rested on theunderlying connective tissue (Fig. 3 A ). Junctional complexeswere observed at higher magnification (arrow, Fig. 3 B ).
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Fig. 2. Scanning electron microscopy (SEM) analysis of bladderuroepithelium from control and SCI animals. Bladders from sham-treatedanimals ( A and B ) or from animals 2 h( C and D ), 24 h ( E and F ), 3 days ( G and H ), 14 days( I and J ), or 28 days ( K-N ) afterSCI were fixed, processed, and then analyzed by SEM. A, C, E, G,I, K, and M, lower-magnification views ofrepresentative areas; B, D, F, H, J, L, and N, higher-magnification views of boxed area in micrograph at left. Arrows in F, small shrunken cellsassociated with epithelium.+ w- J- _8 R) d2 [; L! ^

Fig. 3. Transmission electron microscopy (TEM) analysis of bladderuroepithelium from control and SCI animals. Bladders from sham-treatedanimals ( A and B ) or from animals 2 h( C and D ), 3 days ( E and F ), or 28 days ( G and H ) after SCIwere fixed, processed, and then analyzed by TEM. A, C, E,and G, lower-magnification views of representative areas; B, D, F, and H, higher-magnification views.Arrows, position of junctional complexes in B, D, and H. Distinct junctional complexes were not observed in F. *, Cells that have lost their cytoplasmic density.& T4 X. U" E) O9 w$ C0 v
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The morphology of the uroepithelium was significantly altered by SCI.In addition to normal-appearing areas of uroepithelium, patches ofsmall cells were noted 2 h after SCI (boxed area in Fig. 2 C and higher-magnification view in Fig. 2 D ).These latter areas consisted of disorganized patches of cells that were10-25 µm long and exhibited a relatively smooth surface (Fig. 2 D ). A junctional ring was observed at the periphery ofmany, but not all, of these cells. Because of their small size andmorphology, it is likely that they represent underlying intermediate orbasal cells that were exposed as a result of umbrella cell layerinjury. Analysis of these areas by TEM confirmed disruption of theuroepithelium. Instead of several cell layers, the uroepithelium inthese areas often consisted of only one or two cell layers (Fig. 3 C ). In addition, the cells lining the surface of theseareas sometimes appeared necrotic and lacked an electron-densecytoplasm, apparently the result of plasma membrane disruption. Such acell is shown in Fig. 3, C and D.
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Changes were noted at other time points as well. At 24 h afterSCI, the surface uroepithelium appeared significantly disorganized. Instead of the flat regular appearance of the umbrella cells in controlanimals, the cells lining much of the bladder of these SCI animalsappeared small (10-20 µm long) and had a bumpy, cobblestone appearance (Fig. 2 F ). The surface of the cells wasrelatively smooth. Small shrunken-looking membranous structures(arrows, Fig. 2 F ) were also observed and appeared to beassociated with surface lining cells and might represent necroticuroepithelial cells. At 3-4 days after SCI, a frank hematuria wasnoted in many of the animals. Consistent with this observation, weobserved areas of the uroepithelium that contained denuded or necrotic uroepithelium (Fig. 2 H ). Adjacent areas appeared lessdisrupted. When examined by TEM, these adjacent areas consisted of afew cell layers, and the surface cells often lacked cytoplasmicdensity, appeared necrotic, and had pycnotic nuclei (Fig. 3, E and F ).7 w) m; y& b3 z- _. w( S

However, 2 wk after SCI, the uroepithelium appeared to regenerate. Theepithelium was continuous, had a cobblestone appearance, and consistedof small surface cells (10-20 µm long) with a rough appearance(Fig. 2, I and J ). At 28 days after SCI, theuroepithelium in many animals had significantly recovered. Theepithelium was continuous and relatively flat, and surface folds wereapparent (Fig. 2, K and L ). Although similar tocontrol epithelium, it was not identical, inasmuch as the cells in SCIanimals appeared significantly smaller than in controls and were10-20 µm long. When examined by TEM, the epithelium of the SCIanimals consisted of several cell layers (Fig. 3 G ), andprominent junctional complexes were noted between adjacent cells (arrowin Fig. 3 H ). Some of the 28-day post-SCI animals developedlarge stones in their bladders. The nature of these stones is unknown,but they did not appear to involve bacterial infection, inasmuch as theurine was clear and there was no evidence of cystitis. Theuroepithelium from these animals was morphologically altered and wasreminiscent of 24-h samples. The cells were small, had a cobblestoneappearance, and appeared fairly smooth (Fig. 2, M and N ).
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Effect of hexamethonium and capsaicin on acute (2 h) effects ofSCI. We were surprised to find significant changes in TER and the morphologyof the uroepithelium 2 h after SCI. To better understand theunderlying cause of these acute changes, we assessed whether blockersof neurotransmission could prevent the acute SCI-dependent effects onTER as well as urea and water permeability. Pretreatment withhexamethonium, a blocker of ganglionic transmission, prevented thedecrease in TER observed in 2-h post-SCI animals (Fig. 4 A ). Nosignificant effect on water or urea permeability was noted in theseanimals (Fig. 4, B and C ). The protective effectsof hexamethonium were not observed 24 h after SCI, and the values for TER and urea and water permeability were similar to those ofuntreated SCI animals (data not shown)." h1 N, _! T' f  U5 t8 e, T4 h' r

Fig. 4. Effect of hexamethonium and capsaicin on acute changesin TER ( A ), water permeability ( B ), and ureapermeability ( C ) after SCI. Bladders were obtained fromanimals as follows: control, 2 h after SCI (SCI), pretreated withhexamethonium before SCI (HEX SCI), pretreated with capsaicinbefore SCI (CAP SCI), capsaicin pretreated and then shamoperated (CAP Sham), or injected with PBS beneath the dura of thespinal cord (Needle to cord). Bladders were mounted in Ussing chambers,and TER, water permeability, and urea permeability were monitored. Datafor control and SCI are from Fig. 1. Values are means ± SE from4-32 bladders. * P

Pretreatment with capsaicin, which suppresses C-fiber input from thelower urinary tract ( 9, 11, 43 ), potentiated the effect ofSCI. TER was lower in these animals than in untreated SCI animals (Fig. 4 A ), and there was a significant increase in water and ureapermeability (Fig. 4, B and C ). Vehicle alone had no effect on these treatments (data not shown), and capsaicin treatmentin the absence of SCI had no significant effect on TER or waterpermeability but marginally increased urea permeability (Fig. 4 )., O3 o- n. y2 S3 e9 p1 C. L( H  m
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To evaluate whether acute stimulation of neural pathways may lead tochanges in uroepithelial barrier function, we inserted a needle beneaththe spinal cord dura and injected PBS (in place of lidocaine) into thegray matter. The effect of this acute stimulation or perturbation onurothelial TER, water permeability, and urea permeability was assessedafter 2 h. Acute insertion of a needle beneath the dura alonewithout spinal cord transection caused a significant decrease in TER(Fig. 4 A ) and a significant increase in urea permeability(Fig. 4 C ). However, this treatment had no effect on waterpermeability (Fig. 4 B ).
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SEM analysis was used to assess the effects of these treatments onuroepithelial morphology. The umbrella cell layer of the hexamethonium-treated SCI animals lacked any obvious disruptions (cf.Fig. 5, A and B, with 2-h post-SCI tissue in Fig. 2, C and D ).Continuous junctional rings were observed at the periphery of eachcell, consistent with the TER results described above. When examined byTEM, the cells appeared normal (data not shown). One change was noted:the umbrella cells of these animals were smaller in diameter than thoseof untreated control animals, and their apical surface appeared to behighly wrinkled. The presence of plaques and hinge regions confirmedthat these were umbrella cells. Bladders from hexamethonium- andsham-treated animals appeared normal (Fig. 5, C and D ). The surface architecture of the umbrella cells ofcapsaicin-pretreated SCI animals appeared fairly intact. The umbrellacells were large and had a continuous tight junction band surroundingeach cell, and no discontinuities in the umbrella cell layer wereobserved (Fig. 5, E and F ). However, the surface of the cells appeared relatively smooth, with only a few surface folds.The umbrella cell architecture of sham-operated, capsaicin-pretreated animals appeared normal (Fig. 5, G and H ). Thesurface of umbrella cells from animals in which a needle was insertedinto the spinal cord appeared smooth and resembled the surface ofumbrella cells 2 h after SCI (Fig. 2, C and D ). There were no obvious discontinuities between the cells;however, the diameter of the umbrella cells was highly variable, andpatches of large and small cells were noted.7 V$ C7 h, d5 x; B6 ?" S2 X2 e

Fig. 5. SEM analysis of effects of hexamethonium, capsaicin, and nervestimulation on acute changes in uroepithelial morphology after SCI.Bladders were obtained from animals as follows: pretreated withhexamethonium before SCI ( A and B ), hexamethoniumpretreated and then sham operated ( C and D ),pretreated with capsaicin before SCI ( E and F ),capsaicin pretreated and then sham operated ( G and H ), and efferent nerve stimulated by needle placement intogray matter of spinal cord ( I and J ). A, C,E, G, and I, lower-magnification views ofrepresentative areas; B, D, F, H, and J, higher-magnification views of boxed area in micrograph at left., I) Z% D/ _6 f2 s( A" q0 `

DISCUSSION

SCI is accompanied by a number of well-described changes in normalbladder function: loss of voluntary control of micturition, bladderhyperactivity, bladder/sphincter dyssynergia, development of spinalcord reflexes (including unusual reflexes that are triggered bycold-water instillation or perineum stimulation), increased intravesical pressure, and bladder hypertrophy ( 21, 56 ).In experimental animal models, a hemorrhagic cystitis is often noted early after SCI, and in patients, SCI is well correlated with anincreased incidence of bacterial cystitis and cancer ( 37, 39, 54 ). The effects of SCI on the uroepithelium, which forms thebarrier between the urine and the underlying tissue, are not well understood.
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Disruption of the uroepithelium in response to SCI. Many SCI patients exhibit mucosal ulceration and irritation( 22 ). Using a well-described rat model system, we haveobserved that SCI is accompanied by considerable changes inuroepithelial morphology and barrier function. Within 2 h of SCI,we observed areas of disrupted uroepithelium that lacked surfaceumbrella cells, exposing the underlying intermediate cell layer.Consistent with this disruption, we noted that the TER of theuroepithelium was significantly decreased. Although this decrease couldreflect increased ion transport, we believe that the morphology points to a disruption of tight junctions and cell-cell contact. In contrast, we noted only minor alterations in water and urea permeability. Itcould be that the underlying intermediate cells provide a reasonable barrier to water and urea at this early point after SCI. We observed what appeared to be the formation of tight junctions in the exposed intermediate cells, which is consistent with our recent observations that selective injury of the umbrella cell layer is accompanied by arapid differentiation of the intermediate cell layer, including expression of uroplakin III and formation of tight junctions( 23 ). However, it is possible that the tight junctionsformed at these early stages are not as impermeable to ion flow asthose found in differentiated umbrella cells.0 X. v& w/ H: \# K+ S
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A decrease in TER and a corresponding increase in water and ureapermeability were observed 24 h after SCI. The uroepithelium atthis point lacked umbrella cells in large areas and was generally covered by what appeared to be small intermediate and/or basal cells.By 3-4 days after SCI, the animals often had a hemorrhagic cystitis, which correlated with disruptions in the uroepithelium, significant decreases in TER, and an increase in water and urea permeability. The cause of this cystitis is unknown but is likely toreflect disruptions of the uroepithelium and vasculature and underlyinginflammation provoked by infiltration of urine into the underlyingtissue. Other possible factors are discussed below. By 4 wk after SCI,spinal reflexes were established, the animals could void involuntarily,and TER and water and urea permeability returned to baseline. Thebladder appeared to be hypertrophied during this time, and theuroepithelium appeared generally normal; however, the surface umbrellacells were significantly smaller. This may indicate increased turnoverof surface lining cells.
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Role of efferent nerves and other factors in epithelial injury. Several factors could contribute to the observed changes in the barrierfunction of the uroepithelium. The ability of hexamethonium, aganglionic blocking agent, to prevent the epithelial disruptions andthe corresponding decrease in TER after acute SCI indicate thatefferent pathways arising from the autonomic nervous system could beinvolved in the acute stages of this response. Stimulation of neuralpathways by insertion of a needle beneath the dura of the spinal cordinto the gray matter was also sufficient to change the surfacemorphology of the umbrella cells, decrease TER, and increase ureapermeability. However, the effect was not identical, because disruptionof cell-cell junctions was not observed, indicating that the lesionassociated with neural stimulation was in the apical membrane or tightjunction barrier. Although it is possible that a drop in blood pressureafter spinal transection may contribute to the uroepithelialalterations, this is unlikely, inasmuch as hexamethonium also resultsin a drop in blood pressure, and treatment with hexamethonium beforeSCI reversed the acute effects of SCI. Hexamethonium might act bysuppressing sympathetic or parasympathetic input to the urinary bladder.4 _$ o% d* [0 `9 `& z

Cutting the spinal cord could stimulate neural release of stresshormones, such as catecholamines, which would contribute to theuroepithelial alterations after acute SCI. Altered catecholamine levelsin a number of tissues have been demonstrated to influence mucosalintegrity ( 44, 50 ). Our data have revealed thatintravesical application of norepinephrine can significantly reduceepithelial viability (shown by decrease in TER compared with vehiclecontrols, data not shown). In further support of an effect ofcatecholamines on epithelial function, other studies have shown thatadministration of stressors, such as norepinephrine, results in adesquamation of the bladder urothelium ( 52 ). Although themechanism has not been identified, there is some suggestion that theeffect on mucosal integrity may be due to alterations in the tightjunction, which leads to desquamation of the epithelial cells( 52 ). One possible trigger for these events may be throughsecretion of a chemical mediator from epithelial cells such as NO,which in excess concentrations has been demonstrated to alter mucosalintegrity ( 29, 41, 42, 46 ). We previously showed thatadrenergic agonists, including the, -agonist norepinephrine, canevoke NO release from urinary bladder uroepithelial cells ( 4, 6 ). Although further experiments are needed to establish amechanism for these events, taken together, these data suggest apossible involvement of the sympathetic nervous system in uroepithelialdamage after acute spinal injury.

In addition to the possible release of catecholamines from stimulatedefferent nerves, other factors may contribute to the changes inuroepithelial barrier function. The submucosal tissue underlying theuroepithelium is populated by mast cells ( 12, 25 ).Intriguingly, mast cells in the gastrointestinal tract and in bladdersare intimately associated with neuronal processes and can secrete theircontents in response to neurotransmitters ( 20, 25, 35, 47 ). Thus SCI-dependent neurotransmitter release from efferentnerves could stimulate mast cell release of several mediators,including histamine, bradykinin, prostaglandin D 2,leukotriene C 4, and proteases, all of which couldcontribute to tissue damage and inflammation ( 20, 45 ).These and other inflammatory modulators released from other immunecells might be significant players in the loss of barrier functionnoted at 2 or 24 h after SCI and the cystitis we observed 3-4days after SCI.

Although the breach in barrier function and cystitis observed 1-3days after SCI is likely the downstream consequence of the initialdisruption of the barrier, some of the damage could have also resultedfrom several factors, including overdistension of the bladder, a resultof the loss of voluntary control of voiding. To guard against this, thebladder was manually compressed several times a day after the SCI, andwhen the bladders were excised from animals 1 or 3 days after SCI, theyrarely appeared to be obviously overdistended. However, we cannotrule out that SCI induced some overdistension that may have potentiatedor prolonged the barrier disruption. Although catheterization may havereduced some of these effects, we did not evaluate this procedurebecause of possible inflammatory changes or damage to the urothelium, which might result from placement of the catheter. Moreover,catheterization does not prevent cystitis in human patients( 39 ). Finally, stress associated with SCI could causeincreased catecholamine levels or delay healing as a result of theinability of SCI animals to obtain food and water. The latter wasunlikely to be the case, inasmuch as SCI had no obvious effect on theirconsumption of food or water or their ability to defecate (unpublished observations).
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Possible protective role of afferent pathways. Although activation of efferent pathways may have a detrimentaleffect on barrier function, afferent pathways may have a protective role. The neurotoxin capsaicin was used to evaluate the involvement of capsaicin-sensitive small-diameter afferents (A - and Cfibers) in the changes in ultrastructure and permeability of thebladder mucosa after an acute SCI. These changes were not prevented by capsaicin pretreatment, which desensitizes bladder afferents ( 13, 31 ). Capsaicin pretreatment enhanced the susceptibility of the mucosa to injury by further decreasing the TER compared with untreated SCI animals or capsaicin-treated controls. These results are consistent with reports that suggest a contribution by capsaicin-sensitive nerves,which are sensitive to bladder distension and are located adjacent tothe bladder mucosa, to mucosal protection in various tissues afterinjury or inflammation ( 1, 26 ). Studies suggest that thisprotective effect may be due to substance P/calcitonin gene-relatedpeptide content in capsaicin-sensitive bladder nerves ( 38, 49 ). Although there was no significant difference in the surfacearchitecture between these treated animals and treated controls,dilation of extracellular spaces or alterations in tight junctionsleading to increased permeability may not be evident using the presentmorphological approaches. Thus further studies are needed to examine inmore detail the mechanism for these alterations in mucosal integrity.+ G! v3 S  x( }# V  I

Summary. Our results indicate that, in addition to affecting the detrusor muscleand its innervation, SCI also leads to a rapid disruption of theuroepithelial barrier. This was manifested in a loss of cell-cellinteractions, decreases in TER, and increases in water and ureapermeability. The present understanding is that SCI, which leads toloss of neural activity below the level of the lesion, blocksneurotransmitter release from transected nerves. However, our resultsindicate otherwise. The acute effects of SCI on uroepithelial functioncould be blocked by pretreatment with hexamethonium, indicating thatrelease of neurotransmitters by bladder efferent nerves was at leastpartially responsible for the disruption of the uroepithelium. Inaddition, capsaicin treatment magnified the effects of SCI, indicatingthat capsaicin-sensitive afferent nerves may play a protective role inthe process. Although the neurotransmitters and or inflammatorymediators that disrupt barrier function are not well understood, ourobservations indicate that the nerves that innervate the bladder mayplay an important role in regulating the barrier function of theuroepithelium. By modulating the release of neurotransmitters and/orinflammatory mediators, it may be possible to stem the disruption ofthe uroepithelium that accompanies SCI and other inflammatoryconditions, such as interstitial cystitis.: }( B7 u9 [5 |9 C' x
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ACKNOWLEDGEMENTS4 K0 M) h* ^; d9 A! o* o
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We thank W. C. de Groat for critical comments and suggestions.. v$ ?5 g% z' w! ^+ A( T! q/ e
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