
==== Front
7910918
5452
J Inherit Metab Dis
J Inherit Metab Dis
Journal of inherited metabolic disease
0141-8955
1573-2665

36601751
10.1002/jimd.12587
nihpa2017101
Article
Clinical and pathological characterization of ophthalmic disease in a canine model of mucopolysaccharidosis type I
Nenninger Ariel 1
Ben-Shlomo Gil 2†
Allbaugh Rachel 2
Valentine Bethann 3
Snella Elizabeth 3
Jens Jackie 3
Ellinwood N. Matthew 3
http://orcid.org/0000-0001-7952-8024
Smith Jodi 1
1 Department of Veterinary Pathology, Iowa State University, Ames, Iowa, USA
2 Department of Veterinary Clinical Sciences, Iowa State University, Ames, Iowa, USA
3 Department of Animal Science, Iowa State University, Ames, Iowa, USA
Present address

N. Matthew Ellinwood, National MPS Society, Durham, North Carolina, USA.

† Died October 21, 2020.

Correspondence: Jodi Smith, Department of Veterinary Pathology, Iowa State University, 2724 Vet Med Bldg, 1800 Christensen Dr. Ames, IA 50011, USA. jdismith@iastate.edu
29 8 2024
3 2023
16 1 2023
04 9 2024
46 2 348357
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Mucopolysaccharidosis type I (MPS I) is a rare lysosomal storage disease caused by α-L-iduronidase enzyme deficiency, resulting in glycosaminoglycan (GAG) accumulation in various cell types, including ocular tissues. Ocular manifestations in humans are common with significant pathological changes including corneal opacification, retinopathy, optic nerve swelling and atrophy, and glaucoma. Available treatments for MPS I are suboptimal and there is limited to no effect in treating the ocular disease. The goal of this study was to characterize the clinical and pathological features of ocular disease in a line of MPS I affected dogs, including changes not previously reported. A total of 22 dogs were studied; 12 MPS I were affected and 10 were unaffected. A subset of each underwent complete ophthalmic examination including slit lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry, and ultrasonic pachymetry. Globes were evaluated microscopically for morphological changes and GAG accumulation. Clinical corneal abnormalities in affected dogs included edema, neovascularization, fibrosis, and marked stromal thickening. Intraocular pressures were within reference interval for affected and unaffected dogs. Microscopically, vacuolated cells containing alcian blue positive inclusions were detected within the corneal stroma, iris, ciliary body, sclera, and optic nerve meninges of affected dogs. Ganglioside accumulation was identified by luxol fast blue staining in rare retinal ganglion cells. Increased lysosomal integral membrane protein-2 expression was demonstrated within the retina of affected animals when compared to unaffected controls. Results of this study further characterize ocular pathology in the canine model of MPS I and provide foundational data for future therapeutic efficacy studies.

canine
Hurler
mucopolysaccharidosis
ocular
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pmc1 | INTRODUCTION

The mucopolysaccharidoses (MPSs) are a group of autosomal recessive lysosomal storage diseases found in both humans and animals.1 They are characterized by a deficiency in individual lysosomal enzymes necessary for the degradation of glycosaminoglycans (GAGs). MPS subtypes are characterized by varying degrees of neurological and/or somatic disease that results from pathologic intracellular accumulation of GAGs, causing cellular dysfunction and/or cell death. MPS I (Hurler, OMIM 607014; Scheie, 607 016; and Hurler-Scheie, 607 015 syndromes), is characterized by a deficiency of α-L-iduronidase (IDUA, EC 3.2.1.76) and subsequent accumulation of two GAG subtypes, heparan sulfate (HS) and dermatan sulfate (DS).2 Numerous IDUA genotype variations have been reported but phenotype variations are more limited. Historically, phenotypes have been categorized as Hurler (most severe), Hurler-Scheie (intermediate), or Scheie (attenuated) syndromes; however, it has more recently been accepted that the three phenotypes represent a continuous spectrum where each form is not biochemically or molecularly distinct.

Children affected with Hurler syndrome develop marked cornea clouding, facial and skeletal dysmorphism, developmental delay progressing to neurodegeneration, cardiac disease, and respiratory disease and, when untreated, experience a markedly decreased life expectancy, often dying within the first decade of life.3 Those with Scheie syndrome often have a normal life span and normal intelligence, but have some of the characteristic facial changes, joint stiffness, and more mild respiratory and cardiac disease.3 In the intermediate form, Hurler/Scheie syndrome, children typically have normal intelligence and mild facial changes, but succumb to the disease in their twenties to thirties due to cardiac and respiratory complications.3 Approved treatment options for MPS I patients included hematopoietic stem cell transplant (HSCT) and/or enzyme replacement therapy (ERT) for multisystemic disease, and corneal transplant for ocular disease, all of which have limitations and complications.4 Novel treatments such as gene therapy and substrate reduction therapy are currently being investigated.

Canine MPS I models do not show the genotypic-phenotypic variety of humans as only a single allelic mutation is present, a G to A mutation resulting in a premature stop codon within intron I of the IDUA gene.5 The natural disease course in the canine is characterized by stunted growth, abnormal limb conformation, degenerative joint disease, hepatosplenomegaly, and corneal opacities. Affected dogs typically reach humane endpoints between 1 and 4 years of age.6,7

Reported ocular changes in patients, other than corneal clouding, include retinopathy, glaucoma, and optic nerve swelling and atrophy, yet no such changes have been reported in canine models.6,8 Reported microscopic pathologic changes in the canine model of MPS I are variable. A single report identified vacuolated cells within the retinal pigment epithelium, iris, choroid, sclera, and conjunctiva.6 Another report identified vacuolated cells only in the cornea and sclera but not in other ocular structures.8 Due to variation in the reported pathologic changes, further characterizing the extent of ocular lesions, expansion of the lysosomal storage compartment, and correlating clinical information in this canine model will allow us to better assess treatment outcomes in the future. The objective of this study was to characterize both clinical and histological ocular changes associated with MPS I in a canine model.

2 | METHODS

2.1 | Animals

The MPS I colony originated in a family of Plott hounds which were cross-bred with beagles and other hound dogs.9 This colony is currently maintained and bred at Iowa State University and all animals (unaffected control and MPS I affected) included in this study were derived from this colony. All work was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Iowa State University IACUC. All dogs were diagnosed at birth via PCR for the mutant IDUA allele using methods previously reported.5 Control animals were either heterozygous for the IDUA mutation or homozygous normal. Ocular tissues used in this study were prospectively (n = 12) and retrospectively (n = 10) acquired from other completed studies.10–16 All other ocular tissues were handled and archived according to the same protocol (see histochemistry and histopathology section). A subset of dogs underwent antemortem ocular examinations as a part of this study (n = 12). The natural disease course observed in these animals is such that limb and joint abnormalities as well as liver and cardiac disease develop first at approximately 3–6 months of age, with ocular and neurologic abnormalities developing at approximately 10 months of age.17,18 After approximately 1 year of age these dogs develop progressive corneal opacity and succumb to their somatic disease at approximately 3 years of age.7,9,17,18 Dogs were euthanized by intravenous sodium pentobarbital overdose. Ages and disease status of dogs examined clinically and/or histologically are as follows; 1.1 (n = 2 unaffected, 2 MPS I), 1.2 (n = 1 unaffected, 2 MPS I), 1.4 (n = 0 unaffected, 3 MPS I) 1.5 (n = 1 unaffected, 2 MPS I), 1.6 (n = 0 unaffected, 1 MPS I), 1.7 (n = 1 unaffected, 0 MPS I) 1.8 (n = 1 unaffected, 1 MPS I), and 2.1 (n = 3 unaffected, 2 MPS I) years of age (Table S1).

2.2 | Ocular examination

A complete ophthalmic examination, including slit lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry and ultrasonic pachymetry, was performed on a subset of dogs (n = 12), 6 MPS I affected and 6 controls, by a single board-certified veterinary ophthalmologist (GBS). Intraocular pressure (IOP) was evaluated first in both eyes to avoid altering the corneal surface using a rebound tonometer (TonoVet). Central corneal thickness (CCT) was then measured using an ultrasonic pachymeter (PachPen) placed at a 90°angle to the cornea. Two drops of tropicamide were then placed in each eye for pupillary dilation. After 15 min the eyes were evaluated and a fundic examination was attempted using indirect ophthalmoscopy. If the pupil was not dilated after 15 min, an additional two drops of tropicamide were placed in each eye, with a max of 3 doses within 45 min.

2.3 | Histochemistry and histopathology

All animals (n = 22) were necropsied immediately following euthanasia, and a full set of tissues was collected into 4% paraformaldehyde. One entire globe from each dog was collected into Davison’s fixative for 24 h and then transferred into 70% ethanol. Globes were parasagittally sectioned perpendicular to the posterior ciliary arteries starting 1 mm lateral to the optic nerve. Sectioned globes were dehydrated through graded concentrations of ethanol and embedded in paraffin. Histologic sections of the entire globe were deparaffinized in xylene and rehydrated through graded concentrations of ethanol. Sections were incubated in either hematoxylin and eosin (H&E), alcian blue (AB), or luxol fast blue stains (LFB). For H&E staining, sections were incubated in freshly filtered Mayer’s hematoxylin solution (BBC 5Biochemical, 3580) for 10 min and alcoholic eosin solution (American Mastertech, 6STE0457) for 2 min. For AB staining, sections were briefly dipped in 0.1 N HCl before being incubated at room temperature in alcian blue (Sigma, A3157) stain (pH = 1) for 30 minutes. Counterstain was applied by washing slides in 0.1 N HCl briefly and then incubated for 1 min in nuclear fast red (Sigma, N8002) counterstain. For LFB staining, sections were incubated in 0.1% LFB stain (Sigma, S3382) for 16 h at 56°C. Sections were differentiated individually in 0.05% lithium carbonate (Fisher, L119–500) and 70% ethanol, counterstained with 0.1% cresyl violet acetate solution (Sigma, C5042) for 6 min, and dehydrated through a graded ethanol series, cleared in xylene, cover slipped and examined using a BX-41 Olympus Trinocular brightfield microscope with an Olympus DP73 digital colored camera and cellSens imaging software (v1.15, Olympus Corporation).

2.4 | Immunohistochemistry and semiquantitative analysis

Immunohistochemistry for lysosomal integral membrane protein 2 (LIMP2) was performed to assess lysosomal volume. Antigen retrieval was performed in citrate buffer heated to 65°C (10 mM, pH 6.0) for 30 min on tissue sections. Following antigen retrieval, endogenous peroxidase was blocked in 3% H2O2/tris buffered saline solution for 20 min, followed by endogenous Fc receptor blocking in normal horse serum for 30 min. Sections were then incubated overnight at 4°C with rabbit anti-LIMP2 (PA3–16802, Invitrogen; 1:500). Slides were then washed and incubated with a pre-diluted HRP-conjugated horse anti-mouse/–rabbit secondary antibody (MP7500, Vector Laboratories) for 30 min at room temperature. These sections were then washed and labeled with NovaRED HRP substrate (SK4800, 2Vector Laboratories) according to kit instructions. Sections were then dehydrated through a graded ethanol series, cleared in xylene, and coverslipped, Negative controls consisted of primary antibody omission and were examined to confirm lack of labeling.

Images of four contiguous, non-overlapping fields of retina consisting of two fields on either side of the optic nerve in LIMP2 labeled sections were taken using a BX-41 Olympus Trinocular brightfield microscope with an Olympus DP73 digital colored camera and cellSens imaging software (v1.15, Olympus Corporation). Images were then annotated to exclude retinal pigment epithelium from analysis. The quantification module in Image J software (NIH) was used to determine chromogen in labeled sections as percent total positive tissue. An unpaired t-test was used to compare means using GraphPad Prism (Version 9.3.1).

3 | RESULTS

3.1 | Ocular examination

On ophthalmic examination, 5 of 6 MPS I affected dogs had varying degrees of corneal edema, neovascularization, fibrosis, and mineral/lipid deposits (Figure 1). The sixth and youngest affected dog (1.1 years) had relatively mild corneal changes. In this dog, there was only a small crescent shaped area of edema and no detectable neovascularization or fibrosis. All eyes of affected dogs did not respond, or only partially responded to mydriatic eye drops (1% tropicamide). Their pupils failed to dilate within 45 min after application. Pupils of unaffected control dogs all dilated within 15 min of tropicamide administration. For this reason, and due to impeding corneal opacities, the fundi of all but one affected dog could not be clinically examined. The pupils of the youngest affected dog did dilate; however, dilation was incomplete and went from a resting size of 5 mm pupil diameter to an incomplete dilation of 10 mm. Upon fundic exam of this dog, slight optic disc swelling was noted. Fundic exams of all unaffected dogs were within normal limits.

Ocular pachymetry revealed that 5 of 6 MPS I affected dogs had thickened corneas as compared with unaffected controls (Figure 2). Average thickness of the unaffected canine corneas was 585 μm (+/− 12.7), which is considered normal.19 Average thickness of the affected canine corneas was increased at 774 μm (+/− 213). Conversely, the youngest affected dog had thinner than normal corneas, at 401 μm (OD) and 388 μm (OS).

Intraocular pressure was within the accepted reference range for normal (11–29 mmHg) in both MPS I affected and unaffected dogs.20 However, 4 of 6 affected dogs had a mildly increased average intraocular pressure compared to age-matched unaffected dogs (Figure 3).

3.2 | Histopathological findings

H&E stained histological sections of the globe from 8 unaffected and 10 MPS I affected dogs were examined (Table S1). The corneas of all 10 affected dogs contained keratocytes markedly expanded by fine cytoplasmic vacuolation (Figure 4B). There was also mild neovascularization present in the corneas of all affected dogs. The stroma was moderately expanded by interlamellar clefts and clear space consistent with oedema. The iris of one affected dog contained moderate numbers of round mononuclear cells with similar finely vacuolated cytoplasm. Within the optic nerve meninges, there were spindled cells (presumptive fibrocytes) with cytoplasm moderately expanded by similar clear vacuoles (Figure 4D). Fibrocytes within the sclera were also moderately expanded by clear to lightly eosinophilic vacuoles (Figure 4F).

3.3 | Histochemical staining

Alcian blue (pH = 1) was used to demonstrate the presence of acidic GAGs, and LFB was used as an indirect marker of ganglioside accumulation in histological sections of the globes from 8 unaffected and 10 MPS I affected dogs (Table S1). Inclusions within corneal keratocytes were frequently AB-positive in affected dogs (Figure 5B). AB-positive inclusions were also occasionally noted in scleral fibrocytes. In affected dogs, rare retinal ganglion cells contained LFB-positive inclusions (Figure 5D).

3.4 | Immunohistochemistry

Within a subset of dogs (6 unaffected, 6 MPS I affected) lysosome volume was assessed in the retina using semi-quantitative immunohistochemistry for LIMP2. LIMP2 labeling was observed in the retina of both groups, and localized predominantly to the retinal ganglion cell (RGC) layer and inner plexiform layer (IPL), with scattered labeling in the inner nuclear layer (INL). Within the RGC layer of unaffected animals, there was faint finely granular cytoplasmic immunoreactivity within retinal ganglion cell bodies. In the IPL of unaffected animals, immunoreactivity was minimal with only scattered faint punctate cytoplasmic immunoreactivity. Minimal to no immunoreactivity was noted within the INL of unaffected animals. In MPS I affected dogs there was moderate to intense coarsely granular to punctate cytoplasmic immunoreactivity within retinal ganglion cell bodies. In the IPL of MPS I affected animals, there was moderate, scattered coarsely cytoplasmic immunoreactivity. Scattered faint punctate cytoplasmic immunoreactivity was noted within the INL of MPS I animals as well (Figure 5E,F). Qualitatively, labeling was more intense within the MPS I affected animals. Semiquantitative analysis demonstrated a significant increase in percent LIMP2 immunoreactivity in MPS I affected (5.932 ± 1.184, mean ± SD) versus unaffected (3.098 ± 1.592) controls (Figure 6A), which did not appear to increase with age (Figure 6B).

4 | DISCUSSION

The objectives of the current study were to characterize ocular clinical and histopathological features of MPS I in the canine model. Herein, we have demonstrated pathologic ocular changes consistent with those observed in human patients with MPS I, such as significant corneal clouding, neovascularization, and fibrosis grossly.1 Additionally, there was marked vacuolation consistent with GAG accumulation and lysosomal expansion in the corneal stroma, iris, sclera, and optic nerve meninges on histologic examination. Retinal lesions were more variable, with the accumulation of material consistent with gangliosides within rare retinal ganglion cells, and expanded lysosomal compartments based on LIMP2 immunohistochemistry.

Complete ophthalmic examinations including slit lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry and ultrasonic pachymetry were performed on 12 dogs, as these clinical features had been previously unreported in the canine model. On ocular examination, the affected dogs’ eyes would not dilate or would only partially dilate with the application of tropicamide, even at concentrations higher than the normal dosing. Incomplete pupillary dilation has been documented in other lysosomal storage diseases, such as Gaucher’s disease, due to preiridial fibrovascular membrane formation (PIFM) and anterior synechia as well as accumulation of storage material within tissues of the iris leading to impaired pupillary function.21 Though no PIFM or synechiae were grossly or histologically observed in this study, accumulated material was observed in the iris of 1 out of 6 MPS I affected dog. Additionally, it is possible that these dogs had some degree of subclinical anterior uveitis occurring to explain the resistance to pharmacological dilation, as a recent publication has described anterior chamber flare in some MPS I affected dogs.22 No observable aqueous flare was present and IOPs were within normal limits in the animals evaluated here so uveitis was not suspected. However, the corneas of most affected dogs included in the present study were too clouded for adequate thorough examination of the anterior chamber for subtle aqueous flare. On histopathologic examination, there was no evidence of anterior uveitis, supporting our clinical findings. A sampling of aqueous humor followed by cytologic examination could be performed in the future to look for the presence of inflammatory cells, which may be present in too low a quantity to cause significant histologic lesions. It is also possible that poor pupillary dilation is a problem with the smooth muscle in the iris due to MPS I, as vacuolated cells were noted within the iris of one dog, but more research is required.

In 5 out of the 6 paired age matched dogs, MPS I affected dogs demonstrated increased corneal thickness compared to their unaffected age matched controls. One MPS I affected dog (1.2 years) had significantly thickened corneas compared to its unaffected age match control, while another MPS I affected dog (1.1 years) had thinner corneas than the unaffected age match control. It is important to note that corneal thickness in MPS I affected dogs did not progressively increase with age which contradicts our hypothesis that corneal pathologic changes would be linear and progress with age. Instead, we demonstrated variation in the rate of corneal thickening between age groups (Figure 2). Lysosomal storage diseases are progressive diseases; as the animal ages, increasing amounts of primary as well as secondary metabolic products accumulate within many different cell types.23 Corneal thickening is due to the significant distension and clefting from GAG accumulation in the corneal stroma, which disrupts the very specific collagen arrangement in the cornea causing corneal opacity.24 If there is only one phenotype of a particular LSD, it is reasonable to hypothesize that with aging there would be uniformly increasing corneal thickness, due to additionally accumulated storage material within the corneal stroma. The variation seen here may be due to phenotypic variation similar to what is seen in human patients; however, published research in dogs indicates only one genotype–phenotype having been documented.6,8,9 It is also possible that intrastromal accumulation of storage material is more complicated, and there are other metabolic or inflammatory processes at play.23

Patients with MPS I may develop glaucoma as a complication of disease due to accumulation of GAGs within anterior segment structures, resulting in narrowing of the iridocorneal drainage angle and decreased outflow of aqueous humor.3 In this canine model, we did find vacuolated cells within the iris, ciliary body, and sclera in the anterior segments, similar to what is seen in the human disease, however the iridocorneal angle did not appear to be narrowed or obscured on histology. To assess whether or not glaucoma is a clinical feature in the canine model, intraocular pressure (IOP) was measured as part of the complete ophthalmic examination. Although the IOP’s of the MPS I affected dogs in this study were within normal reference range, values were on the higher end of the reference interval. However, interpretation of tonometry is complicated by the fact that corneal thickness was frequently altered in MPS I affected animals. It has been documented that increased corneal thickness can lead to falsely elevated IOP’s when measured with rebound tonometry.2,25 This would mean that the true IOP in affected dogs may be well within the reference interval, and that elevated IOP leading to glaucoma may not be a feature in the canine model.

Reports of GAG accumulation within the ocular tissues of MPS I affected canines are variable. One study reported GAG accumulation restricted to the corneal stroma and scleral fibroblasts.8 Another reported it within the conjunctiva, cornea, iris, choroid, and sclera.8,26 In this study, we identified GAG accumulation in corneal stromal cells, scleral fibrocytes, optic nerve meninges, and iris based on histopathological exam and histochemical staining with AB. Ganglioside accumulation, a secondary storage product in MPS I, was also demonstrated by LFB staining very infrequently in retinal ganglion cells. Additionally, LIMP2 immunohistochemistry performed on unaffected and MPS I affected eyes showed significantly increased immunoreactivity, primarily within the retinal ganglion cell layer and inner plexiform layer, in affected animals compared to unaffected controls. Morphologically, the cells with increased LIMP2 immunoreactivity are most consistent with retinal ganglion cells or possible retinal astrocytes; however, double label immunohistochemistry or similar methods would be needed to definitively determine cell type. Expansion of lysosomal compartments in numerous visceral, CNS, and ocular tissues, including the retina, has been well documented in patients with MPS I.27,28 The retinopathy described for MPS I is characterized by photoreceptor loss (primarily rod dysfunction determined by electroretinography) along with pathologic changes to the retinal pigment epithelium (RPE) determined on fundoscopy.29 On histopathological examination, it has been described as degeneration of the outer retinal layers as well as fine fibrillary inclusions within the RPE, and fibrillar inclusions as well as multi-membranous inclusions within RGCs.30 In this study, there was no evidence of RPE disease detectable by histology, and fundic examination was not possible in many of the older MPS I affected animals due to significant corneal clouding and poor pupillary dilation. Electroretinography was not performed as part of this study but could be investigated in future studies. Ocular GAG accumulation findings noted here, as well as the involvement of retinal ganglion cells, appear to align with those documented in human patients.

Few therapies have been found to treat both somatic and neurodegenerative components of the disease without significant side effects, and there is currently no ideal treatment for the ocular disease. Systemic ERT as well as intraocular ERT have been found to be ineffective or only partially effective in treating all components of ocular disease such as optic nerve atrophy and corneal clouding.22,31,32 In this canine MPS I model, systemic ERT did not result in significant improvement in corneal disease despite normalization of lysosomal storage in other tissues.22 HSCT has been found to be somewhat effective in decreasing corneal opacification and optic nerve swelling, but it has not been shown to help with retinal degeneration.2,33 HSCT also comes with significant systemic risks such as graft versus host disease and ocular specific side effects such as cataract development, conjunctivitis, keratoconjunctivitis sicca, pseudomembrane formation, and other corneal epithelial defects.2 Corneal transplant has been attempted, however since this does not treat the underlying enzyme deficiency, the transplanted cornea begins to accumulate GAGs over time.34 Recently, intrastromal injection of adeno-associated viral vector gene therapy has been attempted in a canine model of MPS I with promising results including decreased CCT and decreased corneal edema.35 These findings are preliminary and it is important to note that this technique is associated with increased risks of ocular trauma and infection within the cornea and anterior chamber. The results of this study further characterize ocular pathology in the canine MPS I model and provide additional foundational data for future therapeutic efficacy studies.

Supplementary Material

Supplme Mat

ACKNOWLEDGMENTS

The authors would like to thank V. Montgomery for excellent technical assistance. Open access funding provided by the Iowa State University Library.

FUNDING INFORMATION

Animals used in this study were supported by grant R01 NS085381 Patricia I Dickson PI - Neuroimaging and Neuropathology of MPS I. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.

US National Institutes of Health, Grant/Award Number: R01NS085381

DATA AVAILABILITY STATEMENT

An availability of data and materials statement must be provided by all manuscripts from January 1, 2022. Data availability statements should provide information on where data supporting the results reported in the article can be found including, where applicable, hyperlinks to publicly archived datasets analyzed or generated during the study. The data that support the findings of this study are available from the corresponding author upon reasonable request.

FIGURE 1 Gross ocular appearance of an unaffected (A) and MPS I affected (B) dog. (A) Normal healthy cornea of a 2.1-year-old unaffected control. (B) Moderate corneal edema and clouding with localized lipid/mineral deposits and corneal neovascularization in a 2.1-year-old affected dog

FIGURE 2 Mean central corneal thickness in unaffected and MPS I affected dogs. Bars represent individual animals and x-axis is age in years. Nearly all MPS I affected dogs demonstrate increased corneal thickness compared to unaffected age matched controls

FIGURE 3 Mean intraocular pressure in unaffected and MPS I affected dogs. Bars represent individual animals and x-axis is age in years. All dogs had IOP within reference range (11–29 mmHg) for canines at all timepoints

FIGURE 4 Histologic comparison of the cornea, optic nerve meninges, and sclera in unaffected and MPS I affected dogs. Hematoxylin & eosin, 400x. (A) Corneal stroma from a 2.1-year-old unaffected control dog. (B) Markedly vacuolated corneal stroma from a 1-year-old MPS I affected dog. There is also edema causing clefting of the stroma as well as neovascularization (arrowhead). Lightly eosinophilic foamy storage material can be seen within the vacuoles (arrow). (C) Normal meninges and optic nerve from a 2.1-year-old unaffected control dog. (D) Moderately vacuolated (arrow) meninges from a 1-year-old MPS I affected dog. (E) Normal sclera from a 2.1-year-old unaffected control dog. (F) Markedly vacuolated scleral fibrocytes (arrow) in a 1-year-old MPS I affected dog

FIGURE 5 Histochemical and immunohistochemical staining of cornea and retina in unaffected and MPS I affected dogs. Alcian blue (A–B), luxol fast blue (C–D), and lysosomal integral membrane protein 2 (LIMP2) immunohistochemistry (E–F), 400x. (A) Alcian blue (AB) stained cornea from a 1-year-old unaffected control dog. B) AB stained cornea from a 1-year-old MPS I affected dog. Markedly vacuolated stromal cells contain AB positive storage material (arrow). (C) Luxol fast blue (LFB) stained retina from a 2.1-year-old unaffected control dog. (D) LFB stained retina from a 1-year-old MPS I affected dog. Rare retinal ganglion cells contain LFB positive storage material (arrow). (E) LIMP2 immunoreactivity in the retina of a 2.1-year-old unaffected control. (F) Retinal LIMP2 immunoreactivity in a 2.1-year-old MPS I affected dog. There is increased immunoreactivity in MPS I affected retinas, predominantly in the retinal ganglion cell layer (arrow)

FIGURE 6 Mean retinal LIMP2 immunoreactivity in 6 unaffected and 6 MPS I affected dogs of clinical age. (A) Mean LIMP2 immunolabeling is significantly increased (p-value 0.0094) in MPS I dogs compared to unaffected controls. Error bars represent standard error of the mean. (B) Individual animal data. LIMP2 measurements were taken from four regions of the retina. Each bar represents the mean and standard deviation. At all timepoints, LIMP2 immunoreactivity is increased compared to similarly aged controls but does not appear to increase with age. X-axis is age in years

CONFLICT OF INTEREST

Ariel Nenninger, Gil Ben-Shlomo, Rachel Allbaugh, Bethann Valentine, Elizabeth Snella, Jackie Jens, N. Matthew Ellinwood, and Jodi Smith declare that they have no competing interests.

ETHICAL STATEMENT

All work was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Iowa State University Institutional Animal Care and Use Committee. If vertebrate animals have been utilized, documentation of approval from the Institutional Committee for Care and Use of Laboratory Animals (or comparable committee). Iowa State University IACUC protocol approval #7-13-7601-K.

SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.
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