Hypocretin Measurement in an Icelandic Foal with Narcolepsy

Abstract

A 3-month-old female Icelandic foal was presented with a history of swaying, stumbling, and struggling whenever walking out of the stable (Fig 1 and supporting information Video S1). After a few seconds the foal recovered, but additional episodes of collapsing followed. During these episodes, the foal closed its eyes and lack of consciousness was observed. Between the episodes, the owner could not recognize any abnormalities, and the foal had good body condition. Collapsing episodes were first noticed at an age of 4 weeks. The foal was born on pasture and no abnormalities were observed before. Clinical signs remained constant during the whole observation period. Screen capture from a video of the Icelandic foal presenting with narcolepsy but without cataplexy. Full video as supporting information is available in the online version of this article. No clinically relevant findings were detected on physical examination. The neurological examination (ie, mental status, behavior, posture and gait, cranial nerve testing, postural reactions such as hopping laterally with 1 thoracic limb elevated, reflex testing performed in a standing position, withdrawal reflex, extensor carpi radialis reflex) between 2 episodes was normal. Because of episodic weakness and decreased consciousness, the neuroanatomical localization was suspected to be the telencephalon or ascending reticular activating system (diencephalon and medulla). A sleep disorder caused by narcolepsy was assumed. To rule out metabolic and cardiac causes of collapsing episodes, a CBC, serum biochemical profile, urinalysis, and echocardiography were performed. Hematologic examination identified mild leukocytosis (12.96 × 103/μL; reference range, 5.0–10.0 × 103/μL) with lymphocytosis (6.6 × 103/μL; reference range, 1.5–4.4 × 103/μL). Serum biochemistry results include mildly increased γ-glutamyl-transferase activity (52 U/L; reference range, <45 U/L), other liver enzyme activities were within reference ranges. Bile acid concentration was normal (4.9 μmol/L; reference range, <28 μmol/L), and results of urinalysis and echocardiography disclosed no abnormalities. The atropine test was used to diagnose narcolepsy pharmacologically, 0.07 mg/kg atropine sulfatea as an anticholinergic agent was administered IV.1 Atropine administration eliminated clinical signs for the following 24 hours (Fig 2 and supporting information Video S2). Screen capture from a video of the same Icelandic foal after administration of 0.07 mg/kg atropine sulfate. Full video as supporting information is available in the online version of this article. After initiation of general anesthesia with ketamineb (4 mg/kg) and diazepamc (0.5 mg/kg), cerebrospinal fluid (CSF) was collected from the cerebellomedullary cistern with the foal in lateral recumbency. Total nucleated cell count was 1 cell/μL (reference range, <3/μL) with 1 erythrocyte/μL. Total protein concentration of the CSF was 47 mg/dL (reference range, <70 mg/dL). Blood, urine, and CSF were screened to exclude inborn errors of metabolism as described elsewhere.2 Amino acids, organic acids, complex carbohydrates, and purines and pyrimidines were analyzed in urine, serum and CSF by high-performance liquid chromatography (HPLC), gas chromatography, and mass spectrometry, the results of which identified no inherited metabolic disease. The major part of the collected CSF was frozen immediately after collection in liquid nitrogen, stored at −70 °C, and sent frozen to specialized laboratories for further investigation. Different neurotransmitter and hypocretin concentrations were determined in the CSF of the narcoleptic foal and 2 neurological healthy control horses. 3-Methoxy-4-hydroxyphenylglycol (MHPG) as a metabolite of norepinephrine, homovanillic acid (HVA) as a metabolite of dopamine, and 5-hydroxy-indolacetic acid (HIAA), which is derived from serotonin were determined by HPLC and multiamperometric detection.d The concentrations of these neurotransmitters assayed in the narcoleptic foal and the control horses are presented in Table 1. Additionally, hypocretin-1 was determined in the CSF of the horses by a radioimmunoassay.e3 Results of hypocretin assay were 305 pg/mL in the narcoleptic foal, 361 pg/mL in the neurological healthy control foal, and 320 pg/mL in the neurological healthy adult control horse. Three months later, the owners requested euthanasia of the foal because of persistent clinical signs. Brain, lungs, kidney, liver, spleen, and heart were examined histopathologically. A slight multifocal meningeal fibrosis of the forebrain and a slight focal microgliosis in the periventricular white matter of the 4th ventricle at the level of the pons were found. Based on these findings and by exclusion of several diseases, idiopathic narcolepsy without cataplexy was diagnosed in the Icelandic foal. Idiopathic narcolepsy with cataplexy is a very rare neurological disorder, which is described in humans, dogs, and different horse breeds.4-6 Narcolepsy without cataplexy can be observed in Warmblood, Icelandic, and Thoroughbred horses.6 Narcolepsy without cataplexy is characterized by paroxysmal sleep attacks but not accompanied by complete loss of muscle tone and areflexia. The affected foals often remain standing in a trance-like stance and stagger around as observed in this case (Video S1). Affected animals do not become recumbent during the episodes. The pathophysiology of this disease is unknown. Comparison of canine and human narcolepsy suggests a dysfunction of the brainstem, hypothalamus, limbic system, and possibly the striatum and cortex.7, 8 According to this neuroanatomical localization, narcolepsy seems to be a complex biochemical disorder of large parts of the brain with dysfunction of the dopaminergic, cholinergic, and noradrenergic neurotransmitters, which also are involved in the normal sleep-wake cycle and rapid eye movement (REM) sleep.7, 9, 10 Therefore, neurotransmitters of these systems have been evaluated for diagnostic purposes in humans, dogs, and horses (Table 1).8, 11-13 HVA and HIAA as representative metabolites of dopamine and serotonin are low in dogs with narcolepsy, whereas results are inconsistent in humans (Table 1).12 The CSF concentrations of these neurotransmitters in the described narcoleptic foal are comparable with the results of the normal control animals (Table 1). MHPG, which is unaltered in narcoleptic dogs and humans, seemed to be higher in the narcoleptic foal than in the control horses.12, 14 This neurotransmitter is a metabolite of norepinephrine, and adrenergic neurons cease firing in REM sleep.10 Therefore, the increased concentration of MHPG was considered to be because of increased parenchymal turnover of norepinephrine to MHPG. Data on neurotransmitters in foals are lacking and therefore caution must be exercised when comparing results, in particular when the analytical methods differ (gas chromatography and mass spectrometry versus HPLC with amperometric detection) and the preanalytical conditions are not defined. Our method of neurotransmitter analysis (HPLC with multiamperometric detection) is based on that of Heales15 and has been extensively validated for use in children with neurological disorders. The aminergic system includes cells in nuclei that are located in the periventricular gray matter of the mesencephalon and pons (serotoninergic: dorsal raphe nuclei, cholinergic: pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, adrenergic: locus ceruleus). No abnormalities were found in these nuclei on histopathological examination, with the exception of mild microgliosis in the periventricular white matter of the 4th ventricle. However, based on its limited extent, microgliosis was considered to be an incidental finding, unrelated to the observed neurological signs. More recently, the neurotransmitter hypocretin, which is released from the hypothalamus, is used for diagnosis of narcolepsy in humans and dogs.16, 17 To the authors' knowledge, hypocretin has not been assayed in equine narcolepsy cases before, and is reported in the current case for the first time. The sporadic form of narcolepsy is associated with markedly decreased hypocretin concentration in the CSF in humans and dogs (<110 pg/mL; reference, >200 pg/mL).16, 18 Hypocretin concentration in the foal of this report was similar to the concentration in the control horses, healthy dogs and humans, implying that there was no obvious involvement of the hypocretin system in the narcolepsy of this horse at the time of investigation. In the familial form, a mutation in the hypocretin receptor-2 gene causes narcolepsy in the dog.19 Individuals with the familial form of narcolepsy have normal hypocretin concentration in CSF.16, 18 The familial form is documented in humans, dogs and miniature ponies, but not in Icelandic horses.13, 19, 20 Pedigree analysis revealed no cases of narcolepsy in related horses of the foal of this report. A repeated mating of the same mare and stallion in the following year resulted in a normal foal. In humans, where narcolepsy is only rarely associated with mutations in the hypocretin receptor genes, narcolepsy is considered an autoimmune disease, which in most cases is linked with the human leukocyte antigen (HLA).21 Ninety to 100% of patients with definite cataplexy are HLA-DQB1*0602 positive.4, 18, 22 Recent studies suggest that human narcolepsy may be because of the loss of a small number of hypothalamic neurons containing hypocretin and subsequent gliosis in this area of the brain.23 Neurons might be destroyed by an autoimmune reaction.24, 25 At the onset of the disease, only selected hypocretin projections may be involved, which results in sleep abnormalities with normal hypocretin concentration. In the later phase of the disease, complete destruction of hypocretin-delivering cells may occur resulting in undetectable CSF hypocretin concentrations.24 Notably, low concentration of hypocretin so far has only been described in patients in which cataplexy was already present, whereas patients without cataplexy usually have normal concentrations of hypocretin.18 The current case report of a foal with narcolepsy but without cataplexy might reflect initial onset of the disease with normal hypocretin concentration. Hence, narcolepsy without cataplexy may be a preliminary stage of narcolepsy with cataplexy in the horse as is suspected in humans.18 a0.5 mg/mL, Atropinsulfat B. Braun, Melsungen, Germany bKetamin 10%, Medistar, Holzwickede, Germany cDiazepam Ratiopharm, Ulm, Germany dCoulArray, ESA Analytical Ltd, Buckinghamshire, UK eRK-003–30 Orexin A [Human, Rat, Mouse], Phoenix Pharmaceuticals, Belmont, CA The study was not supported by a grant or other funding. Video S1: Icelandic foal presenting with narcolepsy but without cataplexy. Video S2: The same Icelandic foal after administration of 0.07 mg/kg atropine sulfate. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.