Corresponding author: S. Giguère, DVM, PhD, DACVIM, Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Drive, Athens, GA 30602; e-mail: firstname.lastname@example.org.
Background: Neonatal hypoxic-ischemic encephalopathy (NHIE) is a disease affecting newborn foals for which there is no antemortem diagnostic test.
Hypothesis: Ubiquitin C-terminal hydrolase 1 (UCHL1) and the phosphorylated axonal forms of neurofilament H (pNF-H) are markers of brain injury in foals with NHIE.
Animals: Thirty-three foals with a clinical diagnosis consistent with NHIE and 17 healthy foals.
Methods: Retrospective study. Concentrations of UCHL1 and pNF-H in plasma were measured by ELISA. The performance of the assays for the diagnosis of NHIE was assessed by receiver operating characteristic curve analysis. Concentrations of UCHL1 and pNF-H were measured throughout the brains of 2 healthy foals.
Results: The diagnostic performance of UCHL1 (AUC = 0.86) was significantly higher (P= .001) than that of pNF-H (0.52) for the diagnosis of NHIE. Median concentrations of UCHL1 (6.57 ng/mL; 2.35–11.90 ng/mL) in foals with a clinical diagnosis of NHIE were significantly (P < .001) higher than those of healthy controls (2.52 ng/mL; 1.4–4.01 ng/mL). The right sided reference interval for UCHL1 concentrations in healthy foals was 0–4.01 ng/mL. The sensitivity and specificity of UCHL1 (>4.01 ng/mL) for diagnosis of NHIE were 70% (51–84%) and 94% (72–99%), respectively. UCHL1 concentrations were higher in gray than white matter, while pNF-H concentrations were higher in white than gray matter.
Conclusions and Clinical Importance: UCHL1 has potential as a marker of brain injury in foals with NHIE.
Hypoxic-ischemic encephalopathy (also known as neonatal encephalopathy, perinatal asphyxia, dummy foal syndrome, or neonatal maladjustment syndrome) affects neonatal foals. The incidence of the disease in equine neonates is 1–2% of all births.1 Neonatal hypoxic-ischemic encephalopathy (NHIE) in foals is frequently associated with adverse peripartum events such as premature placental separation and dystocia. Very little is known about the pathophysiology of the disease in foals. Work in human neonates and laboratory animals suggest that hypoxia and ischemia initiate a cascade of cellular events that include increased release of excitatory amino acids, calcium influx, loss of ionic gradients, and depletion of energy.2,3 Pathways of inflammation, excitotoxicity, protease activation, and free radical production lead to neuronal cell death.2,3 Clinical signs of NHIE are apparent at birth or develop over the 1st 72 hours of life. The spectrum of clinical signs in affected foals range from mild behavioral abnormalities to severe neurologic deficits including central blindness, coma, and seizure.4 Currently, diagnosis of NHIE is based on clinical signs and exclusion of other differential diagnoses.
An objective marker of equine NHIE allowing detection of neuronal injury would be beneficial for diagnostic purposes. Such a biomarker should be specific to the central nervous system, and provide a sensitive and specific test of neuronal injury. In addition, such a biomarker would be of most practical use if it could be detected in plasma or serum rather than cerebrospinal fluid (CSF). While it is technically much easier to detect neuronal injury biomarkers in CSF, CSF samples are more difficult to obtain and the procedure is relatively invasive.
One potential blood biomarker of neuronal injury is the heavily phosphorylated form of the major neurofilament subunit NF-H (pNF-H). Because pNF-H is found primarily in axons, increased serum concentrations of this protein are expected to provide a specific measure of axonal injury. Serum or plasma concentrations of pNF-H mirror the amount of ongoing axonal loss both in human patients and in various animal models of neurologic disease.5–12 Furthermore, increased CSF pNF-H concentrations are observed in human patients with disorders expected to be associated with axonal loss such as encephalitis, encephalomyelitis, hydrocephalus, subarachnoid hemorrhage, spinomuscular atrophy, multiple sclerosis, and stroke.13
Ubiquitin C-terminal hydrolase 1 (UCHL1) is a more recently recognized biomarker of neuronal injury. UCHL1, previously known as Pgp 9.5, is found primarily in neurons and neuroendocrine cells with a very high concentration in dendrites and perikarya.14,15 Increased plasma concentrations of UCHL1 therefore reflect increased blood brain barrier permeability and destruction of neurons. UCHL1 concentration increases in the CSF of patients with aneurysmal subarachnoid hemorrhage, surgically induced circulation arrest for aortic aneurysm repair, and traumatic brain injury.16–18 The CSF of recovering aneurysmal subarachnoid hemorrhage patients contains large amounts of UCHL1 in a pattern of release broadly comparable to that of pNF-H, as expected if the concentrations of both biomarkers reflect aspects of neuronal loss.19
The objective of the present study was to determine if plasma concentrations of pNF-H and UCHL1 can differentiate between foals with a clinical diagnosis consistent with NHIE and normal foals. Additionally, we sought to evaluate if concentrations of these biomarkers were associated with survival or the severity of the neurologic deficits. Finally we measured the concentrations of both proteins in different regions of the brains of healthy foals.
Materials and Methods
Sample Population and Data Collection
Blood was collected in heparinized vacutainer tubes at time of admission from all the foals <4 days of age admitted to the Hofmann Equine Neonatal Intensive Care Unit of the University of Florida and to Rood & Riddle Equine Hospital between January 2007 and June 2008. Case records of all the foals sampled were examined retrospectively for a clinical diagnosis of neonatal encephalopathy, hypoxic ischemic encephalopathy, perinatal asphyxia, or neonatal maladjustment syndrome. Information obtained from the medical records of these foals included age at admission, mental status at admission (normal, standing but lethargic, recumbent), observation of seizure activity and number of seizures during hospitalization, other neurological signs observed, blood culture results, sepsis score,20 outcome (survival, death, euthanasia), reason(s) for euthanasia when available, concurrent disease process(es), and necropsy results when available.
During the study period, blood was also collected from 17 healthy Thoroughbred or Thoroughbred-Quarter Horse crossed foals within the 1st 4 days of life (median 12 hours, range 4 hours to 4 days). These samples were used to establish the diagnostic value of the biomarkers. Foals were considered healthy on the basis of normal gestation and delivery, thorough physical examination, CBC and plasma biochemistry profile, adequate transfer of passive immunity as assessed by measuring IgGa between 18 and 24 hours of age, and lack of behavioral and neurological abnormalities in the 1st 4 weeks of life. Fresh blood samples were centrifuged at 400 × g for 10 minutes and plasma was frozen at −80°C until assayed. The study was approved by the University of Florida Institutional Animal Care and Use Committee.
ELISA for UCHL1 and pNF-H
The pNF-H ELISA with 2 polyclonal antibodies against bovine pNF-H was performed by a commercially available assayb as described previously.5 Briefly, the samples were added to standard Immulon 4BX 96-well ELISA platesc coated with affinity-purified chicken anti-pNF-H antibodyb and blocked in 1% nonfat milk in Tris-buffered saline (TBS). Plasma was diluted 1 : 2.5 in 2% nonfat milk in TBS plus 0.1% Tween-20 (TBSt), to final volume of 50 μL per well, and each sample was run in duplicate. After 1 hour incubation with shaking at room temperature, plates were washed on a microplate washer and each well was incubated with 100 μL of affinity purified rabbit polyclonal pNF-H antibody at 1 μg/mL in TBSt. After 1 hour of incubation at room temperature with shaking the plate was washed again and each well was then incubated with 100 μL of goat anti-rabbit alkaline phosphatase conjugated at 1 μg/mL in TBSt. After a incubation for 1 hour as before, binding was detected with 1 mg/mL p-nitrophenold in 0.1 M glycine, 1 mM zinc sulfate, 1 mM magnesium chloride, pH = 10.4. After signal development, optical density was measured at a wavelength of 405 nm.e A standard curve was generated using serial dilutions of purified bovine brain pNF-H on each plate.
The UCHL1 ELISA was performed as recently described.19 Briefly, purified monoclonal antibody clone BH7 to UCHL1b was diluted to 1 μg/mL in 50 mM sodium carbonate pH = 9.5 buffer and incubated overnight on Immulon 4 BX plates at 4°C. The plates were blocked in TBS containing 5% nonfat milk for 1 hour at room temperature with shaking. Serum samples were diluted 1 : 2.5 in TBS plus 0.1% Tween 20 and 2% nonfat milk to a final volume of 50 μL per well. Incubation with antibodies was performed as for the pNF-H assay, and bound UCHL1 was detected with affinity purified rabbit anti-UCHL1 at a concentration of 1 μg/mL in TBSt. After further washing, plates were incubated with goat anti-rabbit alkaline phosphatase conjugate.d Signal was developed and measured as described for the pNF-H assay. For each ELISA assay, omission of primary antibody control and also a general background control were included.
Western Blotting and ELISA Quantitation of pNF-H and UCHL1 in Foal Brain
Brains were collected from 2 foals <1 week of age euthanized for reasons unrelated to neurologic disease. Small pieces of tissue ranging in weight from 12 to 175 mg were cut out of the brain, and homogenized in 4 M urea, 0.2 mM PMSF, 1 mM EDTA, 1 mM EGTA, 10 mM Tris-HCl pH = 7.2 at a final concentration of 10 mg/mL, a procedure that allows such samples to be run out on either for western blotting or on the pNF-H ELISA.21 Other samples were dissolved in TBSt at 10 mg/mL containing a protease inhibitor cocktailf for analysis on the UCHL1 ELISA. Regions of both gray and white matter of frontal, parietal, occipital, temporal cortex, regions of the medulla, thalamus, and hypothalamus, the dorsal, medial, and ventral hippocampus, and the corpus callosum were collected from each brain. Samples were centrifuged at 13,000 ×g for 5 minutes to pellet out material that was insoluble in urea or TBS. One microliter of 1 : 100 urea extract or 5 μL of 1 : 10 diluted TBSt extract was loaded per ELISA well for pNF-H and UCHL1 analysis, respectively. An amount equal to 72 μg of wet weight of these extracts was run out in each lane of SDS-PAGE gels. This material was electrophoretically transferred to membranes that were probed with pNF-H and UCHL1 antibodies. Protein assays were performed by the Pierce micro-BCA assay with the brain urea extracts as outlined in the manufacturers manual.g
The overall diagnostic performance of the 2 biomarkers was assessed by use of receiver operating characteristic (ROC) curve analysis.22 Foals with a clinical diagnosis consistent with NHIE were considered true positives and healthy control foals were considered true negatives. The diagnostic performance of the biomarker concentrations in terms of sensitivity and specificity was calculated for each possible cutoff. The area under the curve (AUC) is a summary statistic of overall diagnostic performance of a test. Diagnostic tests can be distinguished as noninformative (AUC ≤ 0.50), less accurate (AUC, 0.50–0.70), moderately accurate (AUC, 0.71–0.90), and perfect (AUC, 1.00; 100% sensitivity and 100% specificity).22
Normality of the data and equality of variances were assessed by the Kolmogorov-Smirnov and Levene's tests, respectively. The data did not meet the assumption for analysis by parametric tests despite attempts at transformation. Continuous variables were summarized as medians (10–90th percentile). The Mann-Whitney U-test was used to test for differences in UCHL1 concentrations between foals with a clinical diagnosis consistent with NHIE and healthy controls, between survivors and nonsurvivors, and between foals with seizures and foals without seizures. Correlation between UCHL1 concentrations and continuous variables such as sepsis score, IgG concentrations, and age was assessed by Spearman's coefficient of rank correlation. The right-sided, 90% reference interval for UCHL1 concentrations in healthy foals was calculated by means of a nonparametric percentile approach. All analyses were performed by a commercial software package.h For all analyses described above, a 2-sided P value <.05 was considered significant.
Plasma was obtained from 33 foals with a clinical diagnosis of NHIE during the study period. The median age at time of sampling was 6 hours (range 0–48 hours). Neurologic deficits noted in these foals included altered mentation (lethargic, obtunded [n = 33]), seizures (n = 12), abnormal nursing behavior (n = 6), and bilateral mydriasis (n = 1). One or multiple concurrent diseases were present in 13 foals, and 20 foals had a diagnosis of NHIE only. Concurrent diseases in affected foals included bacterial sepsis, meconium aspiration, pneumonia, ruptured bladder, rib fractures, hemorrhagic enteritis, renal failure, ruptured gastrocnemius, and jejunal strangulation. Blood culture was positive in 6 of 16 foals sampled. The median sepsis score was 7 (2–13). Seven foals had a sepsis score ≥11. In this study, 2 foals died spontaneously, 7 were euthanized, and 24 were discharged from the hospital. Histopathology of the brain revealed lesions compatible with NHIE in the 3 foals in which it was performed. These lesions consisted mainly of multifocal neuronal necrosis of varying degree of severity in the gray matter of the cerebral cortex, caudate nuclei, thalamus, hippocampus, cerebellar cortex, and medulla oblongata.
To evaluate and compare the diagnostic value of UCHL1 and pNF-H for the diagnosis of neurologic disease consistent with NHIE in foals, plasma concentrations from the 33 foals with a clinical diagnosis of NHIE were compared with those of 17 age-matched clinically healthy foals. Using ROC analysis, the overall diagnostic performance of UCHL1 (AUC = 0.86; 95% confidence interval [CI] = 0.73–0.94; P= .0001) was significantly higher (P= .001) than that of pNF-H (0.52; 0.36–0.68; P= .497) for the diagnosis of NHIE (Fig 1). Median concentration of pNF-H (0.116 ng/mL; 0.107–0.128 ng/mL) in foals with a clinical diagnosis consistent with NHIE was not significantly (P= .512) different from that of healthy controls (0.117 ng/mL; 0.100–0.125 ng/mL). The sensitivity and specificity of UCHL1 for the diagnosis of NHIE at various cutoff points are presented in Table 1. Median concentration of UCHL1 (6.57 ng/mL; 2.35–11.90 ng/mL) in foals with a clinical diagnosis consistent with NHIE was significantly (P < .001) higher than that of healthy controls (2.52 ng/mL; 1.4–4.01 ng/mL) (Fig 2). Median UCHL1 concentration in the 13 foals with NHIE and concurrent disease(s) (5.7 ng/mL; 1.5–29.9 ng/mL) was not significantly different (P= .580) from that of foals with a diagnosis of NHIE alone (6.7 ng/mL; 2.5–11.41 ng/mL). Similarly, median UCHL1 concentration in the 12 foals with failure of passive transfer of immunity (6.8 ng/mL; 1.9–12.1 ng/mL) was not significantly different (P= .472) from that of foals without failure of passive transfer of immunity (6.8 ng/mL; 2.7–31.7 ng/mL). The right-sided reference interval for pNF-H concentrations in healthy foals was 0–0.125 ng/mL. The right-sided reference interval for UCHL1 concentrations in healthy foals was 0–4.01 ng/mL. The positive and negative predictive values of UCHL1 at various disease prevalences for a cutoff of 4.01 ng/mL are presented in (Fig 3). Ten foals with a clinical diagnosis consistent with NHIE had UCHL1 concentrations <4.0 ng/mL. All 3 foals with a confirmed histopathologic diagnosis of NHIE had concentrations >5.0 ng/mL.
Table 1. Sensitivity and specificity of UCHL1 ELISA at selected cutoff points for detection of foals with a clinical diagnosis of NHIE (n = 33) compared with clinically healthy controls (n = 17).
The concentrations of UCHL1 between survivors and nonsurvivors and between foals with or without seizures were not significantly different (P= .856 and .443, respectively). In foals with a clinical diagnosis of NHIE, there was no significant correlation between UCHL1 concentrations and age (r= 0.087; 95% CI =−0.265 to 0.417; P= .418), IgG concentrations (r= 0.172, 95% CI =−0.201 to 0.501; P= .337), and sepsis score (r= 0.310, 95% CI =−0.037 to 0.591; P= .079).
Western blotting and ELISA were used to measure concentrations of both UCHL1 and pNF-H in different regions of newborn foal brains (Fig 4). The lowest concentrations of pNF-H were seen in regions of cerebral cortical gray matter while the highest concentrations were seen in the medulla and midbrain. In contrast, the lowest concentrations of UCHL1 were seen in medulla and other deep brain regions and the highest were seen in the cortical gray matter. The ratio of highest to lowest determination in the foal brain for pNF-H was 26, while that for UCHL1 was 28.6. The highest pNF-H signal detected by ELISA corresponded to 26.6 μg/mg protein in medulla, while that for UCHL1 was 11.1 μg/mg protein in cerebral cortex.
The present study demonstrates that UCHL1 has potential as a biomarker of neuronal injury in neonatal foals with clinical signs of NHIE. Previous studies in other species have provided evidence that CSF concentrations of UCHL1 reflect the severity of neuronal injury in a variety of damage and disease states.16–19 The present study shows that, with a sufficiently sensitive assay, UCHL1 can also be detected in plasma, and that measured concentrations are significantly higher in foals with a clinical diagnosis consistent with NHIE compared with healthy foals. UCHL1 is specific for neurons where it is found in high concentrations. Increased UCHL1 concentrations are indicative of neuronal death. Finally, serum or plasma concentrations can be measured, thereby avoiding the need for CSF collection. In the present study, UCHL1 concentrations did not appear to predict outcome. However, these results should be interpreted with caution as many foals had concurrent life-threatening diseases that might have been the reason for death or euthanasia. These concurrent diseases would not be expected to increase UCHL1 concentrations. In addition, UCHL1 concentrations were only measured on admission in the present study. It is possible that the window for the peak concentrations was missed in some cases. Further studies with larger numbers of foals are needed to determine the dynamics of UCHL1 release with clinical signs of NHIE and if plasma UCHL1 concentrations can estimate the magnitude of CNS injury, predict outcome, and reflect the efficacy of therapy.
The major limitation of this study is the lack of definitive antemortem tests for the diagnosis of NHIE. As a result, the diagnosis of NHIE relied strictly on clinical diagnosis. Postmortem examination confirmed the diagnosis in the 3 cases in which histopathology of the brain was performed. However, it is possible that altered mentation in some foals classified as having NHIE in the present study was the result of a systemic illnesses rather than the result of brain injury. This might explain why some foals with a clinical diagnosis had UCHL1 concentrations within the normal reference range. This scenario might have resulted in underestimation of the sensitivity of the UCHL1 assay in the present study. An additional limitation of the present study is the lack of a group of sick foals with no clinical signs of NHIE. Evaluation of the UCHL1 assay in a prospective trial involving a large number of sick neonatal foals will be required before recommending the test for clinical use to ensure that the assay is specific for neuronal damage and not just a marker for various nonspecific illnesses.
The ROC curve for plasma concentrations of pNF-H revealed little ability to differentiate between clinically healthy foals and foals with clinical signs consistent with NHIE. UCHL1 is primarily localized in neuronal dendrites and perikarya, while pNF-H is primarily localized in axons. pNF-H is less abundant in the cortex compared with the brain stem and spinal cord, and concentrations of pNF-H are highest within the white matter deep within the brain.5 The lack of significant increase in serum pNF-H could be because minimal axonal damage occurred in these foals. Alternatively, it might be because of the fact that lesions primarily affect the gray matter23 as evidenced in 3 foals in the present study. Finally, higher concentrations of pNF-H might be more likely to occur later in time in association with secondary injury resulting in loss of neurons with projection axons. In 1 study, leakage of pNF-H into the blood was more pronounced several days after subarachnoid hemorrhage, and was considered to indicate neuronal loss from delayed ischemic events such as cerebral vasospasm.7 Although these results appear discouraging, it might be valuable to test more foals with NHIE over longer time periods using more sensitive 2nd generation pNF-H assays.10
The data on the concentrations of UCHL1 and pNF-H throughout the foal brain show that pNF-H is heavily concentrated in white matter, while UCHL1 is more heavily concentrated in gray matter. Both proteins are extremely abundant, with pNF-H representing ∼2.5% of the urea soluble protein of medulla while UCHL1 may represent ∼1% of the urea soluble protein of the cerebral cortex, findings in line with previous studies.5,15 Release of UCHL1 in quantity should therefore occur as a result of damage primarily to gray matter, while release of large amounts of pNF-H would indicate damage primarily to white matter. Relatively much more pNF-H was found in deep brain regions such as the medulla and midbrain, while UCHL1 was a more minor component in those regions. The blood UCHL1/pNF-H ratio may therefore give clues about the likely region of brain damage in HIE and other kinds of brain injury.
Early recognition of NHIE is important in human medicine.3 Various biomarkers have been investigated in order to recognize and treat humans with NHIE earlier in the disease process.24 In infants with NHIE, S100β and neuron-specific enolase were significantly increased and were associated with severity of the disease and outcome.25–28 To the authors' knowledge, these markers have not been investigated in foals.
Differentials for a neonatal foal with neurological signs include cerebral trauma or hemorrhage, hydrocephalus, bacterial meningitis, CNS malformations, and septic encephalopathy. In a retrospective study of 84 neonatal foals with signs of neurologic disease, 78 foals were diagnosed with presumptive NHIE, 2 with meningitis, and 4 with hydrocephalus.1 Neurological signs are similar in cases of hydrocephalus, meningitis, trauma, or septic encephalitis. In the aforementioned retrospective study, severe seizures were reported in all 4 cases of hydrocephalus and in 50% of the foals with NHIE. Seizures occurred in 36% (12/33) foals with clinical signs of NHIE in the present study. UCHL1 would be expected to increase in any neurologic disease with significant neuron cell death. Therefore, it would not be expected to differentiate foals with NHIE from foals with other neurologic disorders.
Foals with clinical signs of NHIE are at increased risk for failure of transfer of passive immunity and sepsis. In this study, at least 8 foals had concurrent diagnosis of sepsis. Septic encephalopathy is considered a common complication of sepsis in humans29 and might play an important role in septic neonatal foals. Diagnosis of human septic encephalopathy is contingent on mental status and the presence of an extracranial infection.29 Theories on the cause of septic encephalopathy include the effect of inflammatory mediators on the brain or a cytotoxic response by brain cells to these mediators. The specific effect of sepsis on brain cells would be difficult to disentangle from other physiological problems of sepsis such as hypotension, ARDS, endocrine, or electrolyte imbalances. The resulting pathophysiology involves decreased cerebral blood flow, cerebral edema, and disruption of the blood-brain barrier.29 More work is needed to define septic encephalopathy in foals and to discern if biomarkers will help to differentiate pathological pathways.
UCHL1 warrants further study as a biomarker of NHIE in foals. Longitudinal analysis of UCHL1 concentrations and correlation with clinical severity and outcome will determine its future usefulness as a biomarker of brain injury. Future studies will be required to discern if pNF-H plays a role in NHIE, possibly increasing later in time or in certain disease processes with axonal injury. In the future, a panel of biomarkers may give the most information, with UCHL1 indicating neuronal death, pNF-H reflecting axonal injury, and other biomarkers indicating activation of other brain cells such as astrocytes and microglia.
a DVM Stat, Corporation for Advanced Applications, Newburg, WI
b EnCor Biotechnology, Gainesville, FL
c Fisher Scientific, Pittsburgh, PA
d Sigma, St Louis, MO
e BioTek, Winooski, VT
f P8340 at 1 : 1,000; Sigma
g Thermo Scientific, Rockford, IL
h MedCalc for Windows, version 220.127.116.11, MedCalc Software, Mariakerke, Belgium