Equine laminitis model: Lamellar histopathology seven days after induction with oligofructose

Summary

Reasons for performing study: The histopathology of laminitisduring its transition from the acute to the chronic phase hasnot been previously documented. Studying hoof lamellartissues 7 days after induction of laminitis may provide insightinto the intractable nature of the chronic phase of the disease.

Objectives: To induce laminitis and investigate hoof walllamellar tissues 7 days after dosing.

Methods: Laminitis was induced using oligofructose in 6 normal Standardbred horses. The dorsal hoof lamellartissues of these and 12 normal horses were processed andexamined by light microscopy. Serial sections of a lamellar tipaffected by laminitis were used to create a 3 dimensionalreconstruction.

Results: Transverse sections of dorsal hoof wall lamellae weresignificantly longer than normal. Many secondary epidermallamellae were not connected to primary lamellae and existedas spherical or ovoid, discrete islands isolated in the lamellardermis. The lamellar basement membrane was intact.

Conclusions: Lamellar tissue has the ability to reorganiserapidly following an episode of acute laminitis. Althoughhistopathological evidence of ongoing acute laminitis wasabsent by 7 days, there was marked disruption of lamellararchitecture.

Potential relevance: The architecture and subsequent strengthof the resultant lamellar interface could be greatly influencedfor the better by strategies that minimise mechanicaldisplacement during the acute phase of laminitis.

Introduction

The histopathology of acute laminitis is characterised byattenuation and tapering of lamellae and dysadhesion of thelamellar basement membrane (Pollitt 1996; Johnston et al. 1998;Pollitt and Daradka 1998; Morgan et al. 1999). Normally, lamellartissues are devoid of leucocytes but laminitis promotes an earlyinflux of leucocytes into both the dermal and epidermalcompartments as well as extravasation of red blood cells (Pollitt1996; Morgan et al. 1999; French and Pollitt 2004; Black et al.2006). Lamellae, injured by hoof wall stripping, respondremarkably rapidly and reconstruct an almost normal epidermal

EQUINE VETERINARY JOURNALEquine vet. J. (2009) 41 (8) 735-740doi: 10.2746/042516409X444953

735

Equine laminitis model: Lamellar histopathology seven daysafter induction with oligofructose A. W. VAN EPS and C. C. POLLITT*

Australian Equine Laminitis Research Unit, School of Veterinary Science, Faculty of Natural Resources Agriculture and Veterinary Science,The University of Queensland, St. Lucia, Queensland 4072, Australia.

Keywords: horse; laminitis; chronic; histopathology

architecture if the lamellar basement membrane (BM) survivesrelatively intact (Daradka and Pollitt 2004). On the other hand, 48 h after laminitis has been induced with carbohydrate, much ofthe BM is not only dislocated but lysed (Pollitt and Daradka 1998)and little is known about the fate of the lysed lamellar BM, theseparated BM or the BM still adherent to remaining epidermalbasal cells (EBCs). A hallmark of the histopathology of chroniclaminitis is degeneration and dysplasia of secondary epidermallamellae (SELs) in particular (Morgan et al. 1999). This is markedby the formation of dysplastic epidermal ‘pearl-like’ structuresisolated in the lamellar dermis (Roberts et al. 1980). There ischaracteristic hyperplasia of primary epidermal lamellae (PELs)peripherally (adjacent to the hoof wall) with the production ofproliferative cap horn (cellular arcades and small tubules over theprimary dermal lamellar tips), as well as additional tubular andintertubular horn. The severity of the histopathological changesobserved in the chronic condition tends to correlate with the degreeof lameness and it is probably the strength of the lamellar interfacethat determines prognosis (Morgan et al. 1999).

The histopathological features of acute laminitis 48 h afterinduction with oligofructose (OF) have been described (van Epsand Pollitt 2006) and are identical in nature to those described 48 h after alimentary carbohydrate overload with starch (Pollitt1996). It is important to study lamellar tissue at a time point soonafter this initial acute phase to assess the healing response of tissueaffected by laminitis. A better understanding of the lamellarhealing process after an episode of acute laminitis may explainwhy many horses relapse and may also help in developing betterstrategies for managing laminitis affected feet. In this paper, using

*Author to whom correspondence should be addressed.[Paper received for publication 28.01.09; Accepted 20.04.09]

Abbreviations

BM: Basement membraneDHWDP: Dorsal hoof wall to distal phalanxEBC: Epidermal basal cellsKPELL: Keratinised primary epidermal lamellar lengthOF: OligofructosePDL: Primary dermal lamellaPEL: Primary epidermal lamellaPMN: Polymorphonuclear leucocyte SEL: Secondary epidermal lamellaTELL: Total epidermal lamellar length

EVJ 09-020 van Eps_Layout 1 14/10/2009 13:48 Page 1

the previously described laminitis induction protocol (van Eps andPollitt 2006), the histopathology of laminitis 7 days after inductionwith OF is described.

Materials and methods

Ethical approval

The project was approved by a University of Queensland AnimalEthics Committee (AEC) that monitors compliance with theAnimal Welfare Act (2001) and The Code of Practice for the careand use of animals for scientific purposes (current edition). Allanimals were monitored continuously by the investigators and thehorses were inspected by the Consultant Veterinary Officer to theAnimal Welfare Unit at The University of Queensland at therequest of the AEC.

Animals

Eighteen Standardbred horses (12 geldings and 6 mares) withnormal feet and no lameness were used. Of these, 12 control horses (8 geldings and 4 mares) were obtained from a local abattoir aftereuthanasia by captive bolt. The remaining 6 horses (4 geldings and2 mares) were selected for laminitis induction and were housedand fed in stables for 4 weeks prior to the experiment. These horsesalso served as laminitis induced controls in the accompanyingstudy (van Eps and Pollitt 2009).

736 Equine laminitis model: Lamellar histopathology 7 days after induction

Radiography

Just prior to induction lateral to medial radiographs of the front feetwere made with a standard radiopaque rod, 2 mm in diameter,taped to the dorsal surface of the hoof wall. The distance betweenthe outer hoof wall and the distal phalanx was measured at a pointmid-way between the extensor process and the solar margin of thedistal phalanx. Measurements were adjusted for magnification bystandardising against the measured length of the radiopaque rod.Horses with feet showing radiological evidence of chroniclaminitis were excluded from the study. Additional radiographswere made just prior to euthanasia. Induction of laminitis wasachieved using alimentary overload with the carbohydrateoligofructose (van Eps and Pollitt 2006).

Induction and lameness evaluation

The bolus induction dose of 10 g/kg bwt OF was dissolved in waterand administered by nasogastric tube. The horses also received 10%of the induction dose daily in feed for 3 days prior to administrationof the bolus dose. Two h prior to administration of the bolus dose,72 h afterward and, subsequently, at 12 h intervals, the horses wereevaluated for lameness. All horses were walked toward and awayfrom the observer and circled to the right and left. If no or mildlameness was detected at the walk then the horses were also trottedtoward and away from the observer. The horses were maintained onrubber matting and cross tied in the stall for the initial 72 h periodafter dosing. A limited lameness examination, consisting of circlingthe horse in either direction within the stall, was performed every12 h during this period. Lameness was graded using the systemdescribed by Obel (1948). Horses that were considered Obel grade 3–4 were administered a phenylbutazone/sodium salicylatemixture i.v., 4.5 mg/kg bwt and 1.2 mg/kg bwt, respectively(Butasyl)1 at each 12 h interval until resolution of the lameness toObel grade 2 or less. Following the final lameness examination at168 h (7 days) after the OF bolus dose, all horses were subjected toeuthanasia by overdosing with i.v. barbiturate.

Histology

The fore feet of all 18 horses were removed and processed forhistology (Pollitt 1996). A 10 mm thick midsagittal sample of thedorsal hoof lamellae from each hoof was divided into 3 equalblocks; the proximal block beginning just distal to the extensorprocess of the distal phalanx and the distal block ending justproximal to the tip of the distal phalanx. The resultant proximal,middle and distal lamellar blocks were sectioned transversely,stained with haematoxylin and eosin and periodic acid-Schiff, andexamined using light microscopy.

Fig 1: Photomicrograph of left fore hoof lamellae 7 days after laminitisinduction. Inset, at the same magnification, shows lamellae from normalcontrol horse. The PELs affected by laminitis are longer than normal. Thekeratinised axis of the PELs is relatively unaffected but on either side of itare abnormal columns of partially keratinised epidermal cells(arrowheads). Line A = total epidermal lamellar length (TELL). Line B =keratinised primary epidermal lamellar length (KPELL). PDL = primarydermal lamella. Stain = haematoxylin and eosin.

Fig 2: Photomontage of hoof lamella 7 days after laminitis induction. The majority of SELs are much shorter than normal or fragmented into abnormal,epidermal islands of variable shape and size (arrowed). The islands are not connected to the PEL. On either side of the PELs are abnormal columns ofpartially keratinised epidermal cells (arrowheads). Stain = haematoxylin and eosin.


Measurements on histological sections were made usingimage analysis computer software (Image Pro Plus)2. The totalepidermal lamellar length (TELL) was measured from the base ofeach PEL (at the junction with the stratum medium) to the extentof the associated SEL at the epidermal lamellar tip (Fig 1). Thekeratinised primary epidermal lamellar length (KPELL) wasmeasured from the base of each primary epidermal lamella to thetip of the heavily keratinised axis of the PEL, not includingsecondary epidermal lamellae (Fig 1). Tissue samples from 3 ofthe laminitis-affected feet were serial sectioned (100 sectionseach) and analysed in 3 dimensions using computer software(Voxblast)3 to examine further the morphology of laminitis-affected tissue.

A. W. van Eps and C. C. Pollitt 737

Fig 3: Hoof lamellar tip 7 days after laminitis induction. The majority ofSELs are fragmented into abnormal, variably shaped epidermal islands nolonger attached to the parent lamella. In the inset (of a PAS stainedlamellar tip 48 h after laminitis induction) there is extensive basementmembrane pathology and although many of the SELs appear devoid ofepidermal cells some have survived (arrowhead). Surviving epidermal cellsrepopulate SELs using remnant BM as a template and within 5 days createthe SEL islands of the main picture. Stain = haematoxylin and eosin.

Fig 4: Hoof lamella 7 days after laminitis induction. The basementmembrane bordering fragmented SELs, as well as SELs still connected tothe PEL, is for the most part intact (arrowheads). However, there wereisolated small gaps (arrowed) in the BM of epidermal cell islands adjacentto the PEL. Stain = periodic acid Schiff.

Fig 5: Hoof lamellar tip 7 days after laminitis induction. Most of theepidermal cells that formed isolated BM bound islands resemble the basalcells of normal SELs. A few have dark pyknotic nuclei (arrowed) and thereis the occasional vacuolated cell (arrowhead). There are no mitotic figuresand few white blood cells. Capillaries are present (small arrows) at thebases of the EBC islands. Round aggregates of keratinised cells are presentwithin some of the islands (asterisks). Stain = haematoxylin and eosin.

Fig 6: Three dimensional construct from 100 serial sections of a lamellartip 7 days after laminitis induction. Plane A represents a proximal distal cutand shows the shape of the epidermal islands below plane B. Some of theislands terminate a few microns below plane B and appear lozenge shaped,others are irregularly shaped and extend more deeply. The island arrowedstarts and finishes below plane B and is pear shaped.

Fig 7: Hoof lamellae 7 days after laminitis induction. The bases of PDLsare filled with keratinising epidermal cells. The keratinised axis of thePELs is relatively unaffected but on either side of it are abnormal columnsof partially keratinised epidermal cells (arrowheads). The arcades betweenadjacent PELs are filled with new cap horn. Stain = haematoxylin andeosin.


Statistics

Mean values for KPELL and TELL were calculated for the forefeet of each horse. Results for the laminitis group (n = 6) werecompared to those of the control group (n = 12) using anindependent t test. The effect of section level (proximal, middle ordistal) was analysed within the control and laminitis groups usingrepeated measures one-way ANOVA and Bonferroni post tests.Radiographic measurements were compared between 0 h and 7days using a paired t test in the laminitis group. Results areexpressed as the mean ± s.e. Statistical analyses were performedusing GraphPad Prism version 4.024.

Results

All 6 induced horses developed foot pain characteristic of laminitisby 36 h after the OF bolus. Four of the horses progressed to Obelgrade 3 laminitis between 36 and 72 h, and 2 horses never exceededObel grade 2 laminitis. Administration of phenylbutazoneeffectively ameliorated lameness by one Obel grade. Prior toeuthanasia at 7 days, all 6 had Obel grade 2 laminitis.Radiographically, there was a small but significant increase(P<0.05) in the dorsal hoof wall to distal phalanx (DHWDP)distance in the laminitis group at 7 days (19.16 ± 0.51 mm) whencompared with baseline radiographs (18.25 ± 0.48 mm). Palmarrotation of the distal phalanx relative to the dorsal hoof wall was notdetected in any radiograph.

In the laminitis affected tissue the normal symmetricalorientation of the PELs and secondary epidermal lamellae (SELs)was absent. There was obvious lengthening of the lamellae; theepidermal lamellar tips, consisting of abnormal SELs, extendedaway from the PEL keratinised axis into the dermis (Fig 1). ManySELs had become isolated islands of BM-bound basal andparabasal cells (Figs 2, 3 and 4). Many were no longer connectedto their respective PEL, with some connected by thin strands ofepidermal cells. When examined in 2 dimensions, the islands


appeared circular or oval (Fig 5); however, 3D reconstructions ofthe lamellar islands showed many to be spheroid or disc shapedwith no connection to the PEL in any dimension (Fig 6). There wasno obvious leucocytic infiltration in the control or laminitisaffected tissue.

The BM was tightly adherent to the SELs that were stillattached to the PEL, and also to the SELs that had formedepidermal basal cell (EBC) islands within the dermis. There werea few isolated areas adjacent to the PEL where the BM surroundingthe islands was disrupted (Fig 4). EBCs were of similar size andorientation in the control and laminitis-affected tissue andevidence of proliferation in the form of mitotic figures was scant(Fig 5). Necrotic tissue was absent but the nuclei of some EBCswere pyknotic, consistent with apoptosis (Fig 5). Thrombi were notdetected in either lamellar or sublamellar vessels and erythrocyteextravasation was not observed. In addition there were aberrantcapillary proliferations; e.g. in the centre of lamellar tips (Fig 5).However, small capillaries between SELs appeared reduced innumber; although this was not subjected to detailed analysis.Occasional extravasated polymorphonuclear leucocytes (PMNs)were observed in the control and laminitis samples. In 3 of thelaminitis horses, the distal sections contained columns of partiallykeratinised EBCs (caphorn) present within the tips of the primarydermal lamellae (PDLs) adjacent to the stratum medium (Fig 7).

Within both the laminitis and control groups there was anincrease in KPELL and TELL from proximal to distal. TELL wassignificantly increased in the middle (4372 ± 267 µm) and distal(4513 ± 310 µm) sections compared with the proximal sections(3946 ± 230 µm) in the laminitis feet (P<0.05). In the controltissues, TELL was significantly increased in the distal sections(3622 ± 103 µm) compared with the proximal sections (3237 ± 88 µm) (P<0.05). KPELL did not significantly differ betweensection levels in the control samples. In the laminitis tissue therewas a gradient of severity from proximal to distal; the more distallamellae were more severely affected (Fig 8). There was asignificant increase in TELL in the laminitis tissue compared withthe control tissue at each section level (P<0.05). The most markedincrease was at the middle and distal section levels. Laminitis didnot cause a significant increase in KPELL at any level comparedwith the control tissue. There was a small but significant increasein KPELL in the distal sections (3132 ± 121 µm) compared withthe proximal sections (2806 ± 153 µm) within the laminitisaffected tissue (P<0.05).

Discussion

Seven days after alimentary overload with OF (5.5 days after theonset of acute laminitis) the processes that caused lamellar BMdysadhesion and lysis, basal cell dislocation and lamellarattenuation noted in acutely affected tissue (van Eps and Pollitt2006) appeared to have abated. Almost all the epidermal cells wereenveloped in normal appearing BM and the majority of epidermalbasal cells (EBCs) were of normal shape and orientation. Themajor abnormality was the spectacular change in lamellararchitecture. Normal PEL and SEL anatomy was severelydisrupted. The epidermal strands and islands, many no longerconnected to their respective PEL, had clearly lost their capacity tofunction as a suspensory apparatus between the dorsal hoof walland the distal phalanx. The pathological changes observed 7 daysafter laminitis induction, had markedly reduced the surface area ofthe DHWDP attachment apparatus. The resumption of

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Middle DistalFig 8: Total epidermal lamellar length (TELL) and keratinised primaryepidermal lamellar length (KPELL) for the forefeet of the normal controlhorses (n = 12) and the laminitis horses (n = 6) 7 days after OFadministration, at the proximal, middle and distal section levels (means ±s.e.). There was no significant difference in KPELL between the control andlaminitis sections at any level. The TELL was significantly greater in thelaminitis tissues at each section level (*P<0.05). n = TELL; n = KPELL.


performance activity, and subsequent increased foot loading, couldconceivably rupture the weakened lamellar attachments. This maybe the mechanism behind recrudescent laminitis seen in horses thathave apparently recovered from a primary bout of laminitis(Herthel and Hood 1999).

The quiescent appearance of the lamellar region at 7 dayssuggests that much cellular activity had occurred in the 5 daysfollowing the 48 h time period previously studied (van Eps andPollitt 2006) after OF induction dosing. Lamellar epidermal cellscan repopulate intact BM with extraordinary rapidity; the processwas virtually complete by 5 days after experimental surgical hoofwall stripping (Pollitt and Daradka 2004). The source of the cellswas proliferating EBCs that had survived the trauma of wallstripping. Despite the empty shell-like appearance of acute laminitisBM (Pollitt 1996) some EBCs do survive and, displaying the sameproclivities of EBCs after wall stripping, proliferate and migrateover the remnant BM. However, the BM closes around the formingislands before the new SEL can reattach to the PEL. This results inthe formation of numerous isolated epidermal islands (the ‘pearl-like’ structures of Roberts et al. 1980) at SEL tips, and columns orsheets of EBCs on either side of the PEL keratinised axis in the mid-lamellar region. These EBC columns or sheets may arise becausethe BM is often completely lysed between adjacent SELs leavingwhat BM survives in the dermis to reform around BM free EBCs.

A histological feature of acute laminitis after induction withalimentary carbohydrate overload (Pollitt 1996; Pollitt andDaradka 1998; French and Pollitt 2004) or black walnut extract(Black et al. 2006) is the extravasation of numerous PMNs,especially around lamellar tips where there is extensive BMdysadhesion. Often PMNs are within lamellar compartments.However, by 7 days post induction very few PMNs were observed.suggesting that the chemotactic signals that had attracted theminitially had abated. The administration of phenylbutazone in thisstudy may also have reduced the histological evidence ofinflammation in the lamellar tissue. The analgesic response notedafter phenylbutazone administration implies that inflammationwithin the foot was at least partially responsible for lameness inthis study. Other potential origins of pain in laminitis have beensuggested, including increased submural pressure, tearing ofsubmural tissues, ischaemia, excessive contact between the soleand the distal phalanx and neuropathic mechanisms (Morgan et al.1999; Jones et al. 2007). The small increase in DHWDP distancein this study may be indicative of sufficient displacement of thedistal phalanx to cause pressure between its distal peripheralsurface and the sole. Although minimal histological evidence ofinflammation was present in the lamellar tissue in this study, othertissues (such as the sole) were not examined.

At 7 days post induction there was little evidence of mitosis inany of the EBC layers even in areas where lamellae wereconsiderably thickened. Lamellar widening in chronic laminitis isusually attributed to epidermal hyperplasia (Morgan et al. 1999) butthe lack of mitotic figures among EBCs suggests proliferation wasnot occurring at this time point. A serial study of lamellar histologywould be required to confirm that proliferation of EBCs andparabasal cells occurred prior to the 7 day time point studied here.

In both the control and laminitis tissue, the increased length ofthe epidermal lamellae in the distal sections compared with themore proximal sections was consistent with findings of previousmorphometric studies (Linford 1987; Sarratt and Hood 2005). Afeature of laminitis 7 days post induction was significant lamellarlengthening at all section levels. Lengthening was confined to the

A. W. van Eps and C. C. Pollitt 739

nonkeratinised tips of the PELs as there was no significantlengthening of the more rigid keratinised axes of the PELs.Presumably, after lamellar BM dysadhesion, the isolated BMremains embedded in the lamellar corium and is separated from thePELs when the distal phalanx is weightbearing. Subsequently, theBM remnants are repopulated with EBCs but in the dysplasticarrangement documented here. The small but significant increase indistance measurable on radiographs was a reflection of thestretching and attenuation of lamellae quantifiablehistopathologically. This emphasises the importance of goodquality radiographs to assess the severity of the initial laminitisinsult. A small increase in the DHWDP distance measured onradiographs can indicate the presence of significant architecturalderangement of the lamellae as described here. Early radiographscan be used as a yardstick against which to measure any subsequentexacerbation. The use of high quality magnetic resonance imagingmay provide for an earlier and more accurate diagnosis andprognosis for clinical cases in the future (Keller et al. 2006).

Notable at 7 days was the almost complete repair of lamellarBM and that BM adjacent to EBCs was tightly adherent,suggesting that the BM dysadhesion of acute laminitis (Pollitt1996) is rapidly resolved. This emphasises the importance ofefficacious therapy of the primary disease to limit the impact of thelaminitis triggering process. If the period of dysadhesion is shortand resolution of the BM lesion rapid, effective stabilisation of thedistal phalanx relative to the hoof wall may limit lamellardisintegration. If the forces distracting the distal phalanx awayfrom the hoof wall are neutralised then laminitis may not occur atall. Laminitis triggering factors probably affect other BMsthroughout the body, but unique effects may be seen in the equinefoot because of the high loads experienced by hoof lamellae.

The formation of a lamellar wedge is often described as ahallmark of chronic laminitis (Roberts et al. 1980; Morgan et al.1999). There was no gross evidence that a wedge had formed inthe current study. The sporadic areas of cap horn observed in someof the samples may represent the first stage of wedge formationthat was grossly visible after 20 days in another study (Kuwano et al. 2002).

This study describes the clinical, histopathological andradiographic features of laminitis 7 days after alimentary overloadwith OF. The characterisation of lamellar histopathology describedhere can be used for comparison in future studies evaluatingpreventive and treatment methods for laminitis. Although a serialstudy of lamellar histology following OF induction was notperformed, the most striking finding 7 days after OF induction wasthe lack of histological evidence indicating ongoing inflammatoryor degenerative processes, compared with acute laminitis tissueharvested following an identical induction protocol (van Eps andPollitt 2006). This suggests that lamellar tissue has the ability torapidly reorganise and stabilise once the acute insult has abated.The key to preventing severe chronic architectural changes inclinical cases may lie in the temporal identification of the activephase of lamellar damage. The length and timing of thedevelopmental and acute phases varies widely depending on theinciting disease process (van Eps et al. 2004); biomarkers of activelamellar damage, currently under investigation (Riggs et al. 2007),should help define this critical period. If weightbearing forces andmechanical distraction could be eliminated while the lamellae arecompromised, the results of this study suggest that rapidstabilisation of the lamellar interface occurs, potentially preservingthe lamellar architecture.


Acknowledgements

This project was funded by a grant from the Rural IndustriesResearch and Development Corporation (RIRDC) of Australia.The authors are grateful to the Animal Health Foundation ofMissouri, USA, for their continuing financial support. MichaelLee-Bernstein of Scientific Instrument and Optical Sales isthanked for 3D imaging advice.

Manufacturers’ addresses

1Novartis Animal Health, Pendle Hill, New South Wales, Australia.2Media Cybernetics, Silver Spring, Maryland, USA.3Vaytek Inc, Fairfield, Iowa, USA.4GraphPad Software, San Diego, California, USA.

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Author contributions Both authors contributed to the initiation,conception, planning, execution and writing. Statistics were byA.W.v.E.


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Equine laminitis model: Lamellar histopathology seven days after induction with oligofructose