Measurement of 8‐hydroxy‐2′‐deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia

Abstract

Background

Eight‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a biomarker of oxidative damage evaluated in human neurodegenerative disease, has potential to correlate with postmortem diagnosis of neuroaxonal dystrophy/degenerative myeloencephalopathy (NAD/DM) in horses.

Hypothesis

We hypothesized that 8‐OHdG will be higher in CSF and serum from NAD/DM horses compared with horses with other neurologic diseases (CVSM, EPM) and a control group of neurologically normal horses. We also hypothesized that 8‐OHdG will be higher in CSF compared with serum from NAD/DM horses.

Animals

Fifty client‐owned horses with postmortem diagnoses: 20 NAD/DM, 10 CVSM, 10 EPM, and 10 control horses. Serum and CSF samples were obtained between November 2010 and March 2022.

Methods

Case‐control study using biobanked samples was performed and commercial competitive ELISA kit (Highly Sensitive 8‐OHdG Check ELISA) utilized. Concentration of 8‐OHdG was quantitated in both CSF and serum and compared between groups.

Results

No correlation was established between the measures of 8‐OHdG in serum and CSF and group. CSF median [8‐OHdG] for NAD/DM was 169.9 pg/mL (IQR_25‐75: 67.18‐210.6), CVSM 157.1 pg/mL (IQR_25‐75: 132.1‐229.1), EPM 131.4 pg/mL (IQR_25‐75: 102.1‐193.2), and control 149.8 pg/mL (IQR_25‐75: 113.3‐196.4). Serum median [8‐OHdG] for NAD/DM was 130 pg/mL (IQR_25‐75: 51.73‐157.2), CVSM 125.8 pg/mL (IQR_25‐75: 62.8‐170.8), EPM 120.6 pg/mL (IQR_25‐75: 87.23‐229.7), and control 157.6 pg/mL (IQR_25‐75: 97.15‐245.6). Poisson regression analysis showed no difference established once confounding variables were considered.

Conclusions

Eight‐OHdG did not aid in antemortem diagnosis of NAD/DM in this cohort of horses. At the time of diagnosis horses with NAD/DM do not have ongoing oxidative stress.

Abbreviations

3‐NT

3‐nitrotyrosine

4HNE

4‐hydroxynonenal

8‐OHdG

8‐hydroxy‐2′‐deoxyguanosine

CNS

central nervous system

CSF

cerebrospinal fluid

DM

degenerative myeloencephalopathy

NAD

neuroaxonal dystrophy

F2 isoP

F2‐isoprostane

PBS

phosphate‐buffered saline

ROS

reactive oxygen species

1. INTRODUCTION

The neurodegenerative disease termed neuroaxonal dystrophy (NAD)/degenerative myeloencephalopathy (DM) has become increasingly recognized as a cause of neurologic disease in horses. Diagnosis is based on synthesis of clinical information and exclusion of other potential neurologic diseases, with presumptive cases identified via typical signalment, clinical signs, and diagnostic test results including normal cerebrospinal fluid (CSF) cytology, negative infectious disease test results, and normal imaging studies. ¹ , ² Although recent studies have worked toward diagnosis of NAD/DM in living horses, definitive diagnosis can only be confirmed on postmortem examination of the brain and spinal cord. ¹ , ² In addition to diagnosis, understanding the pathophysiology and etiology of NAD/DM in horses presents additional challenges because of the multifactorial nature of the disease, encompassing both genetic and environmental causes. ³ , ⁴ , ⁵ , ⁶

Based on previous studies, there is a component of abnormal vitamin E metabolism or vitamin E deficiency in addition to a genetic predilection contributing to this neurodegenerative disease. ³ Vitamin E is an essential antioxidant for the central nervous system (CNS), and inadequate vitamin E status because of either nutritional deficiency or abnormal metabolism is anticipated to increase oxidative damage in the CNS. ⁷ , ⁸ In some situations, horses might have adequate intake and normal metabolism but still have increased oxidative stress because of environmental factors. Insecticides, wood preservatives, and dirt lots are risk factors for disease. ⁹ Because evidence of oxidative stress is detected on postmortem examination of horses diagnosed with NAD/DM, studies have investigated its contribution to the pathogenesis and etiology of this disease. ¹ Markers of oxidative stress, including lipid peroxidation biomarkers in tissue, have been evaluated with increases detected in certain cholesterols and oxysterols in the spinal cord of horses with NAD/DM. ¹⁰

Various biomarkers including 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), F2‐isoprostane (F2 isoP), 4‐hydroxynonenal (4HNE), and 3‐nitrotyrosine (3‐NT) have been studied in humans with neurodegenerative disease and recently implicated in equine neurodegenerative disease based on similarities in disease process. ¹⁰ , ¹¹ , ¹² , ¹³ In horses F2 isoP, ¹⁰ 4HNE, ¹ and 3‐NT ¹ are identified in tissue suggesting the presence of oxidative damage within the central nervous system (CNS) as a feature of NAD/DM, but a CSF or serum‐based biomarker assay for oxidative damage has not been described. ¹ , ¹¹ 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a commonly used biomarker of oxidative damage, is an oxidized derivative of deoxyguanosine that is produced secondary to DNA damage. ¹⁰ 8‐OHdG has been studied extensively in humans with neurodegenerative diseases such as Alzheimer disease and Parkinson disease to better understand the diagnosis, treatment, and disease progression. ¹⁰

Since neurodegenerative diseases display many similarities across species, our goal was to investigate 8‐OHdG as a potential diagnostic biomarker that correlates with the histopathologic diagnosis of NAD/DM in the horse. The specific objectives of the study were (a) to optimize and validate a competitive ELISA previously reported in equids ¹⁴ for measuring 8‐OHdG in equine serum and CSF and (b) to determine if measurable 8‐OHdG in serum and CSF could aid in the antemortem diagnosis of NAD/DM. We hypothesized that 8‐OHdG will be increased in serum and CSF from horses with NAD/DM compared with horses with other neurologic diseases, including cervical vertebral stenotic myelopathy (CVSM) and equine protozoal myeloencephalitis (EPM), and a control group of neurologically normal horses (control). Additionally, we hypothesized that 8‐OHdG will be increased in CSF compared with serum from horses with NAD/DM.

2. MATERIALS AND METHODS

2.1. Sample population

This case‐control study was performed using biobanked CSF and serum samples from horses with NAD/DM, CVSM, EPM, and control horses. Samples were obtained between November 2010 and March 2022 from client‐owned horses presenting to New Bolton Center for clinical evaluation, with control horses presented for clinical evaluation and subsequent euthanasia. Ethical approval was given by owners for banking of samples. Fifty horses were tested: 20 horses with NAD/DM, 10 horses with CVSM, 10 horses with EPM, and 10 control horses. Diagnosis and placement into 1 of the 4 groups was based on confirmed EPM diagnosis using SnSAG2/4/3 ELISA (Equine Diagnostic Solutions LLC, Lexington, Kentucky) serum : CSF titer ratio and postmortem examination, with CVSM and NAD/DM based on characteristic histopathologic lesions. Control horses had a baseline neurologic examination performed and were less than a grade 1 on the modified Mayhew ataxia scale. ³ Postmortem examination of the CNS was normal for all control horses with complete postmortem results available for all NAD/DM, CVSM, and EPM horses. Banked serum samples were obtained as part of routine clinical assessment or immediately before euthanasia. Banked CSF was available for all horses included. CSF was obtained via atlanto‐occipital or lumbosacral approach. Atlanto‐occipital CSF centesis was performed under general anesthesia as part of routine clinical assessment, or immediately postmortem. Lumbosacral CSF centesis was performed as part of routine clinical assessment. The samples were stored in 1 to 2 mL aliquots within a −80°C freezer until analysis.

Clinical information (Table S1) retrieved from the records of each horse included signalment (age, breed, sex), ataxia score (modified Mayhew scale), serum vitamin E concentration, CSF nucleated cell count, CSF red cell count, CSF total protein, final diagnosis, and date of sample collection and storage.

2.2. ELISA optimization and validation

A commercially available competitive ELISA kit, Highly Sensitive 8‐OHdG Check ELISA (Fukuroi, Shizuoka, Japan), was used to measure 8‐OHdG in paired serum and CSF samples from 50 horses. Initial analysis was performed using the manufacturer protocol (Figure 1). To ensure interpretable data, a single dilution was added to the standard curve to expand the range of the ELISA. The protocol used in the published paper (modified protocol) ¹⁴ was tested (Figure 2), as this varied from manufacturer instructions, before validation of the ELISA. The modified protocol produced unreliable results based on a poor standard curve obtained from the assay. Volume matrix testing was performed which revealed that the volume of standard affected the standard curve but, following a 1 : 4 dilution, CSF matrix did not affect standard dilution, producing an acceptable standard curve. Spike and recovery experiments with CSF and serum matrices were run. Based on volume matrix testing, the ELISA was then optimized using increasing volume of samples, with a maximum of 200 μL per well. The standard was diluted 1 : 4 for final testing of both CSF and serum matrix using 200 μL per well as determined from volume matrix testing. Inter‐ and intra‐assay repeatability were established as 14.3 and 17.8, respectively, and a dynamic standard curve was obtained with a wide range for CSF (Figure 3). The method of sample quantification was asymmetric sigmoidal 5PL. The final optimized ELISA protocol yielded a linear range of the assay between 31.25 and 10 000 pg/mL. ELISA validation was performed using serum and CSF from horses with NAD/DM and the kit standard, resulting in reliable standard curves obtained for each.

FIGURE 1.

Standard curve obtained from running initial CSF and serum samples from horses with NAD/DM using manufacturer instructions before addition of a dilution. Linear range of the assay between 125 and 10 000 pg/mL.

FIGURE 2.

Standard curve obtained from running CSF and serum samples from horses with NAD/DM using manufacturer instructions (4°C) versus modified protocol ¹⁴ (37°C) including additional dilution added for a dynamic range between 31.25 and 10 000 pg/mL.

FIGURE 3.

Standard curve obtained using optimized and validated ELISA for 8‐OHdG. Volume of sample utilized in optimized protocol was 200 μL per well, incubated at 4°C, with addition of dilution with lowest limit of detection being 31.25 pg/mL.

The validated, optimized ELISA protocol was utilized for sample analysis. Samples were brought to room temperature (20°C‐25°C) for analysis. Microcon‐10 kDa centrifugal filter units were soaked for 12 hours. Filters were spun at 9700 rpm for 20 minutes then at 3000 rpm for 3 minutes following rotation (flipping) of the filters. Samples were pre‐treated by filtration following filter preparation using 350 μL of sample (CSF or serum), and subsequently spun at 9700 rpm for 50 minutes. The primary antibody was reconstituted per manufacturer protocol and 50 μL added to each well. Exactly 200 μL of sample or standard were then added to each well, with samples run in duplicate per manufacturer recommendation. The outermost wells along each side of the plate were left blank as recommended by the manufacturer. The plate was covered ensuring a tight seal before incubation at 4°C overnight.

After incubation the contents were poured off and the plate was washed 3 times using 250 μL of phosphate‐buffered saline (PBS) per well, which was discarded. Reconstituted secondary antibody (100 μL) was applied to each well and incubated at room temperature for 1 hour, with the washing procedure repeated. Enzyme substrate (100 μL) was added to each well. Incubation for 15 minutes at room temperature in the dark was performed. Then 100 μL of the terminating solution was added before reading the absorbance at 450 nm using a Spectramax M2E microplate reader (Molecular Devices: San Jose, California). Standards at 31.25, 62.5, 125, 250, 500, 1000, 4000, and 10 000 pg/mL were used to generate the standard curve.

2.3. Statistical analysis

All analyses were conducted with Stata 16.1MP (StataCorp) with 2‐sided tests of hypotheses and a P value <.05 as the criterion for statistical significance. A 2 means t‐test power analysis was performed for the number of animals needed per group with a minimum of 8 per group required based on a 2‐fold increase between populations, similar to what has been documented in human studies utilizing the same biomarker. A preliminary exploratory analysis using Spearman rank correlation was performed. All continuous data were assessed for normality using Shapiro‐Wilk statistic. The data were not normally distributed. Non‐normally distributed data were analyzed with Kruskal‐Wallis population rank test. Poisson regression analysis was performed to assess confounding factors including breed, sex, group, and vitamin E concentration. Linear regression was performed with NAD/DM horses as the reference group for both CSF and serum, with results corrected for age, breed, and sex.

3. RESULTS

Initial results from the manufacturer and modified ¹⁴ ELISA approach resulted in unreliable values based on intra‐assay variability, with poor standard curve, and values below detectable range. Dilution of the lowest standard in conjunction with ELISA optimization allowed for 90% to 92% of samples to have measurable 8‐OHdG concentrations. ELISA validation was performed to achieve a reliable and reproducible standard curve. No difference in standard curve or concentrations of 8‐OHdG were observed following alteration in the standard volume used.

A total of 50 paired serum and CSF samples were utilized for the final analysis following ELISA optimization. There were a variety of breeds included in the study: 25 Warmblood horses (50%), 20 non‐Warmblood horses (40%), and 5 horses without breed information available (10%). A total of 17 Warmblood horses (68% of the total number of Warmblood breed horses) were in the NAD/DM group (85% of the total group). The average age of horses included in the study was 8 years (range 1‐18 years). Four horses had values of 8‐OHdG that were not detectable in serum (below the limit of 31.25 pg/mL) and 5 had values that were not detectable in CSF. A total of 17 NAD/DM, 9 CVSM, 10 EPM, and 10 control horses' serum samples (92%) and 16 NAD/DM, 10 CVSM, 10 EPM, and 9 control horses' CSF samples (90%) had detectable 8‐OHdG concentrations following ELISA optimization and were able to be included in the data analysis. Exactly 18 of the 20 NAD/DM horses had vitamin E concentrations reported, with 2 EPM horses having vitamin E concentrations recorded. Age of the sample based on the date of CSF and serum collection was not available for 6 horses, all of which were a part of the control group. CSF total nucleated cell count, total protein levels and amount of red blood cells were available for 21 of the 50 horses sampled.

Spearman rank correlation showed no significant association between measures of 8‐OHdG in serum and CSF or age of the horse, age of the sample, and the results. No statistical significance was associated between [8‐OHdG] and group, or association with a diagnosis of NAD/DM, CVSM or EPM, based on the Kruskal‐Wallis population rank test. Poisson regression was performed, showing no significant difference once confounding variables (breed, age, age of sample, and vitamin E concentrations) were considered (P > .05 for all comparisons). There was no difference in concentrations of 8‐OHdG amongst disease states (Figure 4; P > .05).

FIGURE 4.

8‐OHdG concentration ([8‐OHdG]) for paired CSF and serum samples collected from horses with NAD/DM, CVSM, EPM, or the absence of neurologic disease (control). Black line indicates median value for each disease state.

Linear regression for both CSF and serum was performed with the NAD/DM group as the reference. CSF median [8‐OHdG] for NAD/DM was 169.9 pg/mL (IQR_25‐75: 67.18‐210.6), CVSM 157.1 pg/mL (IQR_25‐75: 132.1‐229.1), EPM 131.4 pg/mL (IQR_25‐75: 102.1‐193.2), and control 149.8 pg/mL (IQR_25‐75: 113.3‐196.4). Serum median [8‐OHdG] for NAD/DM was 130 pg/mL (IQR_25‐75: 51.73‐157.2), CVSM 125.8 pg/mL (IQR_25‐75: 62.8‐170.8), EPM 120.6 pg/mL (IQR_25‐75: 87.23‐229.7), and control 157.6 pg/mL (IQR_25‐75: 97.15‐245.6). No correlation was established between the measures of 8‐OHdG in serum and CSF.

4. DISCUSSION

After optimization and validation of a commercially available ELISA, 8‐OHdG was detectable in equine CSF and serum. However, it is not a suitable diagnostic biomarker in cases of NAD/DM in this cohort of horses. Upon analysis of confounding variables, no statistical significance was established between increase in this biomarker and detection of disease state or a predictor of a postmortem diagnosis of NAD/DM. No correlation was established between concentrations of 8‐OHdG detected in serum and CSF.

8‐OHdG is an in vivo biomarker of oxidative damage extensively studied in Alzheimer disease, amyotrophic lateral sclerosis (ALS), and Parkinson disease. ¹¹ , ¹² , ¹³ , ¹⁵ Each of these disease states has in common oxidative damage as a suspected underlying etiology. 8‐OHdG is produced in response to oxidative damage, and it is used to measure the rate of DNA damage and repair. Studies have looked at the presence of this biomarker in humans with neurodegenerative disease, control groups, and those with other causes of systemic oxidative damage. ¹² Compared with other causes of neurologic disease aside from neurodegenerative disease, myopathies, and those that are healthy, this biomarker is persistently elevated both in CSF and serum of people with neurodegenerative disease. These findings suggest that it could be more sensitive for oxidative damage within the CNS than elsewhere in the body. ¹²

Although oxidative stress likely plays a role in the etiology of NAD/DM in the horse, its cause, timing, and contribution in NAD/DM remain uncertain. Cases of NAD/DM have been hypothesized to have a genetic link along with suspected vitamin E deficiency or oxidative imbalance early in life. ³ , ⁹ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ 8‐OHdG is identified as a marker of oxidative damage but it is unknown whether this compound remains elevated in the CNS over time or is cleared after the initial insult, as increased concentrations are primarily documented in people with progressive neurodegenerative disease, during which continued production of 8‐OHdG would be expected. ¹¹ , ¹² , ¹³ , ¹⁵ Our hypothesis that 8‐OHdG would be elevated in cases of NAD/DM, in comparison to other disease states, was based on the premise that this disease is a progressive neurodegenerative disease, similar to those in humans, with continued oxidative damage occurring. Given the findings it is possible that the pathophysiology of NAD/DM in the horse revolves around an early insult, genetic predisposition, or a combination as discussed in previous studies, versus a continued process of oxidative damage. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ , ⁹ , ¹⁰ , ¹⁶ , ¹⁷

Providing access to green pastures in foals has been discussed as a potentially protective mechanism against EDM. ⁹ Low vitamin E and limited access to pasture within the first year of life has been discussed in multiple studies as an underlying etiology. ³ , ⁹ , ¹⁶ , ¹⁸ , ¹⁹ , ²¹ When accounting for vitamin E concentration, no difference in 8‐OHdG was identified. Without continued oxidative damage it could be that 8‐OHdG would not be found in high concentrations in the CNS of horses with NAD/DM.

When considering the other neurologic conditions in comparison to NAD/DM, such as CVSM, there is ongoing damage within the CNS that can explain the increased concentration of 8‐OHdG. In cases of CVSM, inflammation within the CNS has been hypothesized because of chronic or intermittent compression/stenosis and subsequent recurrent trauma. ²² , ²³ Inflammatory markers and leukograms were not available for horses in the control group. As a result, the extent of systemic inflammatory response that each horse was experiencing and whether ongoing oxidative damage could be expected remains unknown. Based on studies in humans, it was expected that systemic inflammation independent of the CNS would only affect the serum values of 8‐OHdG. ¹² , ¹³ Systemic inflammatory markers are not elevated in cases of EPM. ²⁴ Because diagnosis of EPM likely lags behind initial infection, we cannot rule out early systemic inflammation as a part of the disease pathogenesis that can lead to an increase in circulating reactive oxygen species (ROS) and oxidative damage. ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸

8‐OHdG was measured in horses diagnosed with asthma because of the proposed pathogenesis involving oxidative damage. Compared with a group of healthy horses, those with asthma have elevated concentrations of 8‐OHdG using the same commercial ELISA kit utilized in this study. ¹⁴ Despite the use of the same ¹⁴ commercial ELISA kit there was trouble replicating the results and achieving measurable concentrations of the biomarker. The previous study used a methodology that varied from the manufacturer instructions, with exact methods not detailed (ie, whether dilutions and further optimization were needed or subsequent validation performed). ¹⁴ Despite attempting to replicate both methods, concentrations remained below the detectable range of the ELISA before optimization. The reason behind this is uncertain; we considered whether age of samples could have contributed, but no clinical significance was detected between age of sample and [8‐OHdG] in this study (P > .05). After optimization and validation of the ELISA, detectable values were obtained. The control population of this study had higher concentrations of 8‐OHdG than those reported in the study of horses with asthma. ¹⁴ The reason for this, aside from a hypothesis of methodology difference, is unknown. Variability in systemic inflammation could contribute, as complete blood cell counts and acute phase proteins were not available in our cohort of horses, and were not discussed in the article. ¹⁴ In humans it is expected that systemic inflammation independent of the CNS would only affect the serum values of 8‐OHdG, although this could have contributed to the findings. ¹² , ¹³

Limitations of this study reflect that of a retrospective case‐control study, with complete clinical history of some of the horses being unavailable. Despite calculation of sample size, not every adjunctive diagnostic test result was available for each horse, which affected the data set and statistical analysis. The importance of vitamin E concentration on [8‐OHdG] was unable to be evaluated for groups aside from those with NAD/DM. The role of 8‐OHdG in horses versus humans remains unknown. If suspected to be associated with active and ongoing oxidative damage it makes sense that other causes of neurologic disease that have an active ongoing process such as CVSM and EPM could have higher measurements of 8‐OHdG than NAD/DM. The lack of studies utilizing this biomarker, or similar biomarkers of oxidative damage in horses, remains a limitation and precludes its use in clinical practice in the horse.

Other markers of oxidative damage or other metabolites within equine CSF might prove more helpful than 8‐OHdG as a diagnostic tool. Given its utility in diagnosis of human neurodegenerative disease, before discarding 8‐OHdG as potentially helpful diagnostic tool, additional studies utilizing a younger group of at‐risk horses with inadequate vitamin E status to see if this group has increased concentrations might provide insight to the pathogenesis behind NAD/DM. In addition, there are a variety of ways to measure 8‐OHdG other than that of a commercial ELISA which could provide results that differ from this study.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF‐LABEL ANTIMICROBIAL DECLARATION

Authors declare no off‐label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Ethical approval was granted for the purpose of this study as the samples obtained were obtained for clinical use and banked for research. Postmortem examinations performed per owner and insurance request.

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.

Supporting information

Data S1. Supporting Information.

Palmisano M, Kulp J, Bender S, Stefanovski D, Robinson M, Johnson A. Measurement of 8‐hydroxy‐2′‐deoxyguanosine in serum and cerebrospinal fluid of horses with neuroaxonal degeneration and other causes of proprioceptive ataxia. J Vet Intern Med. 2024;38(2):1207‐1213. doi: 10.1111/jvim.16988