Absence of Glaucoma in Tg-MYOC^Y437H Mice of Diverse Genetic Backgrounds

Abstract

Purpose

Mutations in the myocilin (MYOC) gene cause elevated intraocular pressure and glaucoma. To better understand the factors influencing susceptibility to glaucoma, we studied the MYOC mutation (MYOC^Y437H).

Methods

We characterized ocular phenotypes in Tg-MYOC^Y437H mice on nine different mouse strain backgrounds.

Results

No glaucoma-related phenotypes were observed. We detected neither elevated intraocular pressure (daytime readings) nor optic nerve degeneration differences between wild type (WT) and Tg-MYOC^Y437H mice. This included an absence of Tg-MYOC^Y437H–induced phenotypes on a mixed B6SJL background that best reflected the original publications with this strain. We confirmed that this result was not due to an absence of transgene expression in ocular tissues.

Conclusions

Our data indicate undefined complexity and that the previously reported glaucoma phenotypes are not robust across all institutions and environments.

Mutations in the myocilin gene (MYOC) are known to cause both juvenile glaucoma and adult-onset primary open-angle glaucoma (POAG).^1–4 These mutations lead to elevated intraocular pressure (IOP), a key risk factor for glaucoma. The mutations compromise aqueous humor drainage from the eye by damaging trabecular meshwork (TM) cells, which are a key component of the ocular outflow pathway.^5–7 Close to 100 different pathogenic MYOC mutations have been identified, and the associated disease phenotypes vary in severity and age of onset. In addition, there is variable penetrance and expressivity of disease associated with individual MYOC mutations,¹ suggesting that genetic background and/or other factors have an important influence on outcomes. Pathogenic mutations primarily cluster in the protein's olfactomedin domain, but the significance of this mutation distribution is unclear.² Various observations in humans and animal studies of myocilin indicate that various mutations act via a gain-of-function.^8–13 Mutations elevate IOP by driving protein misfolding and impeding secretion of MYOC from the cell, resulting in intracellular accumulation of MYOC in TM cells.¹⁴ Although intracellular accumulation alone is not sufficient to elevate IOP in mice,¹⁵ it is suggested to lead to the progressive death of TM cells. MYOC mutations can also impact the morphogenesis of the ocular drainage tissues.^4,13,16

A commonly studied pathogenic mutation in MYOC causes a Y437H substitution in the protein's olfactomedin domain. This MYOC^Y437H mutation is associated with relatively severe disease outcomes, with patients commonly developing IOP elevation and glaucoma in the second decade of life.¹⁷ Mice that express the human MYOC^Y437H mutant protein in TM cells have been reported to develop elevated IOP and glaucoma similar to patients with POAG.^8,18 However, certain genetic backgrounds with the MYOC^Y437H mutant protein have proven to be resistant to IOP elevation and glaucoma.¹⁸ In addition, mice with the equivalent mutation in their endogenous Myoc locus (Myoc^Y423H) across four different strain backgrounds do not develop IOP elevation and glaucoma, despite impeded secretion of the mutant protein.¹⁵ Together, these results indicate that there are various unknown factors (e.g. genetics and environmental) that influence susceptibility to IOP elevation and glaucoma in MYOC^Y437H mutant models.

To further interrogate mechanisms underlying IOP elevation in this POAG model, we imported mice with a transgene expressing the MYOC^Y437H allele (Tg-MYOC^Y437H).⁸ On the original mixed genetic background (C57BL/6J × SJL/J mix, henceforth called B6SJL, see the Methods section), we did not detect glaucoma-associated phenotypes in Tg-MYOC^Y437H mice. As we and others have shown that strain background can have a major impact on IOP and glaucoma phenotypes,^18–20 we decided to transfer the mutation to genetically diverse strain backgrounds. We hypothesized that altering strain background may reveal a robust glaucoma phenotype in Tg-MYOC^Y437H mice. To test this, we analyzed the Tg-MYOC^Y437H on nine different strain backgrounds, including the segregating B6SJL strain background on which this allele was originally published.⁸ Surprisingly, we did not detect any glaucoma-related phenotypes in Tg-MYOC^Y437H mice on any examined background. These results show that the Tg-MYOC^Y437H allele does not robustly produce glaucoma phenotypes in all laboratories.

Methods

Animal Husbandry and Ethics Statement

We imported B6SJL;Tg(CMV-MYOC^Y437H)/Vcs mice as a generous gift from Dr. Val Sheffield's laboratory (University of Iowa). The Tg(CMV-MYOC*Y437H)#Vcs (Tg-MYOC^Y437H) allele was generated by cloning the human MYOC gene into the mammalian expression vector pCS⁺ and a mutagenesis kit was used to introduce the Tyr437His mutation.⁸ The expression cassette containing a CMV promotor and MYOC cDNA was injected into the pronucleus of freshly fertilized C57BL/6J x SJL/J (B6SJL) oocytes.⁸ Founder males were mated to C57BL/6J mice before being intercrossed to generate B6SJL;Tg(CMV-MYOC^Y437H)/Vcs mice.⁸

Although the original genetic background that we received was randomly mixed, we refer to it as B6SJL to reflect its genetic components from both the C57BL/6J and SJL/J strains. We harvested sperm from B6SJL mice and used it to in vitro fertilize C57BL/6J and DBA/2J oocytes. This generated two separate F1 cohorts (called B6xB6SJL F1 and D2xB6SJL F1 mice). We also directly backcrossed the MYOC^Y437H allele from B6SJL mice to the A/J (AJ, Stock# 000646), C57BL/6J (B6J, Stock# 000664), C3A/BLiA-Pde6b⁺/J (C3A, Stock# 001912), and SJL/J strain backgrounds (SJL, Stock# 000686) for at least 6 and up to 11 generations. The Tg-MYOC^Y437H allele was also backcrossed to strain DBA/2J (Stock# 00671) for 8 generations before crossing to the non-glaucomatous DBA/2J-Gpnmb⁺/SjJ substrain (D2-G, Stock# 007048) for 3 additional generations and generated a Gpnmb^+/+ experimental cohort. In addition, we backcrossed the Tg-MYOC^Y437H allele to the C57BL/6NJ-Crb1^rd8+em1Mvw/MvwJ (B6N, Stock# 022521) background, which contains a corrected rd8 allele, for 10 generations. Additionally, we maintained the mutation on the original mixed genetic background by intercrossing and analyzing B6SJL mutant mice at intercross generations 1 to 7. No differences in phenotypes were found between any of these B6SJL intercross generations, so we combined all mice for analysis. As a positive glaucoma control, we included data from 12-month-old DBA/2J (Stock# 00671) mice collected over an overlapping timeframe as Tg-MYOC^Y437H mice. DBA/2J mice develop robust IOP elevation and glaucoma.^21–23 Given ongoing negative results, our move to a new institution, and the coronavirus disease (COVID) pandemic, some backgrounds were only analyzed for IOP or otherwise analyzed in less depth (e.g. B6N).

All of the mice in our experiments were maintained on NIH 31 (6% fat) diet ad libitum with HCl acidified water (pH 2.8–3.2). Tg-MYOC^Y437H and wild type (WT) littermates were housed together. All mice were housed with pine wood shavings as bedding and covered with polyester filters, as previously described.^22,24 Cages were maintained in an environment kept at 21°C with a 14-hour light: 10-hour dark cycle. Cohorts included a balanced number of male and female mice. All mice were treated in accordance with the Association for Research in Vision and Ophthalmology's statement on the use of animals in ophthalmic research. Live animal experiments were conducted at the Jackson Laboratory. The Institutional Animal Care and Use Committee of The Jackson Laboratory approved all experimental protocols. Data generation from harvested samples, data analyses, and manuscript preparation were performed at Columbia University.

Genotyping of the Tg-MYOC^Y437H Allele

The genotype of the Tg-MYOC^Y437H transgene was determined using a PCR protocol. Genomic DNA was PCR amplified with a forward 5′-CGTGCCTAATGGGAGGTCTAT-3′ and reverse 5′-CTGGTCCAAGGTCAATTGGT-3′ primer set specific for the Tg-MYOC^Y437H allele. Genomic DNA was PCR amplified using the following program: (1) 93°C for 3 minutes, (2) 93°C for 15 seconds, (3) 63°C for 1 minute, (4) 72°C for 45 seconds, (5) repeat steps 2 to 4 for 35 times each, and (6) 72°C for 5 minutes. Then, 5 µL of sample was run on a 3% agarose gel. The Tg-MYOC^Y437H allele amplifies a 453-base pair fragment.

IOP Measurement and Anterior Chamber Depth

IOP was measured during the light part of the day using the microneedle method, as previously described.^25,26 Mice were acclimatized to the procedure room and anesthetized via an intraperitoneal injection of a mixture of ketamine (99 mg/kg; Ketlar, Parke-Davis, Paramus, NJ, USA) and xylazine (9 mg/kg; Rompun, Phoenix Pharmaceutical, St. Joseph, MO, USA) immediately prior to IOP assessment, a procedure that does not alter IOP in the experimental window.²⁶ During each IOP measurement period, eyes of independent WT B6 mice were assessed in parallel with experimental mice as a methodological control to ensure proper calibration and equipment function. For the B6/B6SJL and D2/B6SJL F1 cohorts, IOP was measured at 6, 9, and 12 months of age, with all mice within a 2-week age window around those ages. For all other background strains, IOPs were measured at a young timepoint (3–4 months of age), an intermediate timepoint (6–8 months of age), and an older timepoint (12–14 months of age). All cohorts included a balanced number of male and female mice. We compared IOP distributions within all strain backgrounds by 2-way ANOVA, with age and Tg-MYOC^Y437H genotype as independent variables. IOPs of B6N background mice were only compared at one age (12–15 months) and across Tg-MYOC^Y437H genotype. Differences between genotype groups were compared by Tukey's honestly significant difference test. We measured IOP of typically 30 to 50 eyes and at least 25 eyes per group (age, genotype, and strain background) except for SJL background mice at 12 to 14 months where 12 transgenic and 13 WT eyes were examined, and B6N background mice where 53 transgenic and 12 WT eyes were examined.

Anterior Chamber Examination

As anterior chamber depth is a sensitive indicator of exposure to high IOP in mice,^20,27,28 even if the pressure is high at a different time or day or was elevated at an earlier age, we also evaluated the depth of anterior chamber in all eyes. Anterior eye tissues were examined approximately every 3 months between 3 and 15 months of age. Using a slit-lamp biomicroscope, we evaluated all mice for phenotypic abnormalities associated with IOP elevation, including pupillary abnormalities, generalized corneal haze, buphthalmos, and deepening of the anterior chamber, in 10 to 20 eyes from Tg-MYOC^Y437H mice on AJ, D2-G B6J, and C3A backgrounds at 12 to 14 months of age. Only female mice were used for slit-lamp examination. In addition, mice of all nine genetic backgrounds had their anterior chambers evaluated under a dissection microscope using drop PBS on the cornea to provide a clear view into the eye while performing IOP measurements (same ages as above). No differences in phenotypic abnormalities were observed between the methods.

Optic Nerve Assessment

Intracranial portions of optic nerves were dissected, processed, and analyzed as previously described.^29–32 Briefly, optic nerve cross-sections were stained with para-phenylenediamine (PPD) and examined for glaucomatous damage. PPD stains all myelin sheaths of a healthy axon, but differentially darkly stains the myelin sheaths and the axoplasm of sick or dying axons. This allows for the sensitive detection and quantification of axon damage and loss. Significant optic nerve axon loss was previously reported at 4 to 5 months in Tg-MYOC^Y437H mice, with substantial loss by 12 to 14 months.⁸ As we detected no abnormal anterior chamber depth or IOP phenotypes in our mice, we analyzed optic nerves primarily at ages ≥ 12 months (with the majority at ∼ 12 months with a limited number at older ages out to 17 months) for both WT and Tg-MYOC^Y437H mice of all examined backgrounds. As glaucomatous neurodegeneration was not detected at any age, we combined data for all ages.

Degree of damage was assigned using a damage rating scale that is well-validated against axon counting.^30,33 Multiple sections of each nerve were considered when determining the damage level. One of three damage levels was assigned:

• (1)No damage/glaucoma (NO) – less than 5% axons damaged and no gliosis. This level of damage is seen in age- and sex-matched non-glaucomatous mice and is not due to glaucoma.
• (2)Moderate damage (MOD) – average of 30% axon loss and early gliosis.
• (3)Severe (SEV) – > 50% axonal loss and damage with prominent gliosis.

For each background and group, we analyzed 18 to 34 nerves except for C3A and D2 WT mice (n = 17 and 12, respectively) and SJL mice. As SJL mice naturally develop retinal degeneration as a consequence of the Pde6b^rd1 mutation,³⁴ and the effects on glaucoma are uncertain, we restricted examination to a small cohort of 6 Tg and 8 WT nerves. All examined F1 B6SJL mice were either heterozygous or WT for the Pde6b^rd1 allele and so did not develop retinal degeneration. We discarded a small number of both WT and Tg nerves of the B6SJL F1 background from analysis because of very severe segmental damage with swelling resembling ischemia, possibly due to potential stroke and inflammatory risk in this background or some unknown trauma that may have occurred.^35,36 Because of a laboratory move and the lack of detected anterior chamber deepening and IOP elevation, we discontinued examining B6N background mice without examining the nerves. Age- and sex-matched WT nerves of all other genetic backgrounds were analyzed as above.

Optic Nerve Axon Counting

We selected four backgrounds (AJ, B6J, B6SJL, and C3A) for axon counting using the AxonJ software in ImageJ, as previously reported.³⁷ This software enables reproducible axon counting from PPD-stained optic nerve images by applying edge detection, morphological filters, and Hessian-based convolution to distinguish axons from the background. The method has been extensively validated against expert manual counts, demonstrating high accuracy and reproducibility. We randomly selected 3 high-quality sections (technical replicates) per nerve and counted axons within one large rectangular region per section with each rectangle sampling ≈ 50% of the nerve area. These sections were imaged using a Keyence BZ-X810 microscope using the brightfield settings and a 100 × 1.45 NA oil objective. We used AxonJ to count axons twice per section to mitigate seed point variation. To calculate total axons per section, we extrapolated the axon count within each rectangular region to the total area of the nerve in each section and averaged the two measurements for each section. The total axon count for all three sections was then averaged to produce the axon number per nerve. We counted axons for at least 9 and up to 19 nerves for any experimental genotype group, with matched numbers of mice of each genotype belonging for any strain background. Nerves were randomly selected from each group. A small number of randomly selected nerves were not appropriately stained to be reliable for automated counting and were replaced. The number of axons was compared by ANOVA followed by Tukey's honestly significant difference test.

MicronIV and Image-Guided Optical Coherence Tomography

Optical coherence tomography (OCT) paired with the MicronIV: Retinal Imaging Microscope (Phoenix Research Labs) was used to assess the retina and optic nerve head in vivo. Mice were subject to pupillary dilation with a drop of 1% solution of cyclopentolate hydrochloride ophthalmic solution (Akorn, Inc.) that was topically applied to the cornea. Mice were anesthetized as previously described (see the Methods section, IOP measurement). Mice were secured on a rotating stage and imaged using OCT per the manufacturer's directions. From all tested backgrounds, we examined at least 8 eyes per genotype at 10 to 12 months of age. B6xB6SJL F1 and D2xB6SJL F1 cohorts were analyzed prior to OCT equipment being available. Due to a laboratory move and lack of IOP elevation, the B6N background cohort was not examined.

RNA Isolation, cDNA Synthesis, and RT-PCR

We examined expression of the mouse Myoc and human Tg-MYOC^Y437H alleles in eyes of each genotype and strain background between 3 and 7 months of age. For whole eye tissue, fresh postmortem enucleated eyes minus the lens were analyzed. The lens was removed after making a small incision in the cornea. All remaining tissue was immediately placed in RNA later solution (Ambion). RNA was isolated and processed for RT-PCR using the PureLink RNA Mini Kit (ThermoFisher). Total RNA was reverse transcribed using the Verso 1-Step RT-PCR Kit ReddyMix, with ThermoPrime Taq (ThermoFisher). A portion of the cDNA was then used in three separate PCR reactions to detect endogenous mouse Myoc (mMYOC), Tg-MYOC^Y437H (hMYOC), and Gapdh as a control. For mMyoc, we used a primer set: forward 5′-GCCATCCAAGACCTTCAGAG-3′ and reverse 5′-AGATCCCTGGTTTGGGTCTC-3′. For Gapdh, we used a primer set: 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. For hMYOC, we used a primer set: forward 5′-GAGTAAGGCAAGAAAATGAGAATC-3′ and reverse 5′-CCTCTCCACTCCTGAGATAGC -3′. For mMyoc and Gapdh, cDNA was amplified using the following program; (1) 94°C for 3 minutes, (2) 94°C for 30 seconds, (3) 60°C for 30 seconds, (4) 72°C for 1 minute, (5) repeat steps 2 to 4 for 39 times, and (6) 72°C for 5 minutes. The hMYOC cDNA was amplified using the following program: (1) 94°C for 3 minutes, (2) 94°C for 30 seconds, (3) 53°C for 1 minute, (4) 72°C for 1 minute + 1 second/cycle extension, (5) repeat steps 2 to 4 for 39 times, and (6) 72°C for 5 minutes. Then, 5 µL of sample was run on a 3% agarose gel. The mMyoc allele amplifies a 206 base pair fragment, the hMYOC allele amplifies a 249 base pair fragment, and the Gapdh allele amplifies a 452 base pair fragment. We analyzed a total of 12 mice of each genotype across all genetic backgrounds (24 eyes). Despite random variability in the amount of amplified product, the results clearly identified expression of the mutant transgene in all Tg-MYOC^Y437H eyes.

Results

No Detectable IOP Elevation or Glaucoma in Tg-MYOC^Y437H Mice

We first longitudinally characterized IOP in Tg-MYOC^Y437H and WT control mice (lacking the MYOC transgene) of all nine strain backgrounds. We detected no IOP changes between Tg-MYOC^Y437H mice compared with WT mice (Figs. 1A–C, Supplementary Fig. S1). In addition to IOP measurements, we inspected eyes by slit-lamp, confirming the lack of abnormal anterior segment phenotypes in Tg-MYOC^Y437H eyes (Figs. 1D, 1E). Importantly and confirming the IOP findings, the anterior chamber depth was normal in all mice at all ages at which IOP was assessed. This is important because increases in anterior chamber depth are a sensitive and indelible indicator of even modestly elevated IOP in mice.^20,27,28 This allows detection of eyes that have been exposed to high IOP even if the IOP elevation occurred at a different time of day or night to the IOP measurement or occurred at a different age or timepoint. Finally, we examined the optic nerves and retinas of WT and Tg-MYOC^Y437H mice (Figs. 2A–D). For all strain backgrounds, Tg-MYOC^Y437H caused no glaucoma, with no significantly different degree or occurrence of damage beyond non-MYOC^Y437H related differences (age- and or other strain-dependent damage) that were present in WT mice (see the Methods section). This lack of damage is seen in both OCT imaging and in histological examination of optic nerve cross sections. Finally, we counted axon numbers, detecting no significant differences between WT and Tg-MYOC^Y437H nerves.

Figure 1.

IOP in Tg-MYOC^Y437H mice with different genetic backgrounds. (A–C) Boxplots of IOP (interquartile range and median line). Tg-MYOC^Y437H genotype did not significantly change IOP (age- and strain-matched, all P > 0.06, even SJL at 12–14 months which was lowly sampled, P > 0.5). (D) In agreement, no anterior chamber deepening was found in Tg-MYOC^Y437H mice across backgrounds. Age-matched DBA/2J (D2, established glaucoma model) data are shown as a positive control for pathogenic AC deepening. There were 12 to 20 eyes examined in each Tg-MYOC^Y437H group. (E) Examples of AC depth severities with the deep AC image of a positive control eye.

Figure 2.

No significant optic nerve health changes in Tg-MYOC^Y437H mice. (A) Frequency histogram of optic nerve damage evident in PPD-stained cross sections of 10 to 17-month-old nerves (majority 12–14 months, number of nerves examined shown in figure). Twelve-month-old DBA/2J (D2) mice, an established glaucoma model, show a high incidence of severe glaucoma. Moderate damage, likely due to stresses with age, occurs in some nerves of WT and Tg-MYOC^Y437H eyes (Fisher's exact test, all P > 0.3) that were not detected to have high IOP or deep anterior chambers, whereas the subset analyzed had no optic nerve excavation by OCT. Although not significantly convincing between Myoc genotypes, there was a trend to a greater occurrence of this damage in the reasonably sampled A/J and C3A strain backgrounds, but this difference could be due to chance sampling. Deeper sampling would be required to determine if this is due to genotype or chance. (B) Representative images of PPD-stained optic nerve cross sections for different severity levels. Scale bar = 100 µm. (C) Boxplots (interquartile range and median line) of the total number of axons in Tg-MYOC^Y437H and WT optic nerve cross sections. There were no significant changes in axon numbers between genotypes (all P > 0.6). The majority of nerves counted were 12 to 14 months and were age-matched across genotypes. AJ nerves were typically analyzed at an older age compared with other groups (14 months). (D) Representative SD-OCT images of the retina and optic nerve head at 10 months of age. WT and Tg-MYOC^Y437H eyes of each strain background have a normal nerve head. Scale bar = 100 µm. SD-OCT, spectral-domain optical coherence tomography.

Loss of Tg-MYOC^Y437H Expression Is not Responsible for the Lack of Glaucoma Phenotypes

Because no IOP or glaucoma phenotypes were detected despite our analyzing a large number of mice with diverse genetic makeups, we decided to confirm that MYOC^Y437H transgene was still being expressed in ocular tissues. On each strain background, expression of the Tg-MYOC^Y437H transcript was readily detected in mutant but not in WT mice (Supplementary Fig. S2).

Discussion

In our colony and environment, the Tg-MYOC^Y437H allele did not cause either high IOP or glaucoma on any of the nine genetic backgrounds (including F1s) that were aged and analyzed. This is despite our confirming that the mutant gene is expressed and has not undergone some form of spontaneous inactivation. It remains possible that the transgenic allele has changed in some way to explain the differences. However, this seems unlikely as to account for the lack of glaucoma on all strain backgrounds it would either: (1) have had to happen very early in the alleles history, as we obtained the mice prior to the initial publication that introduced it; or (2) it would have had to independently change on different tested genetic backgrounds as independent founder mice were used to establish colonies on different genetic backgrounds. Although independent changes on each of multiple backgrounds seem unlikely, it may be that there was a single common change if the founders shared a common ancestor in which the allele had changed soon after its generation at Iowa and prior to shipping to us, and that these “changed” mice were then not used to propagate the strain at Iowa, but we cannot assess that. Further arguing against the above changes, an independent group recently reported no IOP or aqueous humor drainage abnormalities in Tg-MYOC^Y437H mice separated from the original colony at a later date.³⁸

Although we did not detect glaucoma or increased axon loss compared with strain matched controls in Tg-MYOC^Y437H nerves, we did find age-related damage on all backgrounds, distributed across WT and Tg-MYOC^Y437H genotypes. C3A, AJ, and SJL Tg-MYOC^Y437H eyes had a higher proportion of age-related damage compared with WT nerves (but SJL had very low sample sizes, all P > 0.3). Although not statistically significant, it is possible that the transgene is influencing age-related or other pathologic changes not related to high IOP on these backgrounds, but much deeper sampling would be needed to determine this. In addition, it remains possible that glaucoma-associated phenotypes could be detected on additional untested strain backgrounds or with further aging in our environment. Another laboratory has independently failed to detect the glaucoma-inducing high IOP in Tg-MYOC^Y437H mice.³⁸ The discrepancy in phenotypes between laboratories strongly argues that the transgene does not produce glaucoma in all settings. The reasons for this are not clear and must involve environmental factors, as we have included genetic contexts as close as possible to those previously reported to develop glaucoma. The nature of the important environmental factors remains unknown, but the presence or absence of specific gut bacteria alters other conditions (e.g. rheumatoid arthritis, asthma, and type 2 diabetes).^39–41 Aligned with possible microbial differences, our mice were housed with acidified water that modulates microbial growth in their water bottles and could alter the microbiome. However, acidified water was not used in an independent study that also reported no changes in IOP, aqueous humor outflow facility (a direct and sensitive measure of ocular drainage tissue function), or TM cell density (used on 6–7-month-old Tg-MYOC^Y437H mice of a B6SJL F1 background).³⁸ If the microbiome is important, differing rederivation and housing conditions between institutes could alter the phenotypes. We are aware that other groups have not detected glaucoma but have not published their findings due to the inertia against publishing negative results, so it is not clear how commonly these mice fail to develop high IOP and glaucoma at different institutions. The type of diet and how it is sourced and treated, the type, treatment, and changing frequency of mouse bedding, the rate of cage air exchange/type of cage, and many other variables could have an impact. Substantial work would be required to resolve this.