Evaluation of endothelin-1 and MMPs-2, -9, -14 in cerebrospinal fluid as indirect indicators of blood–brain barrier dysfunction in chronic canine hypothyroidism

Highlights

• ET-1, MMP -9 and -14 do not appear to play a significant role in BBB damage that occurs in dogs with chronic hypothyroidism

• CSF may not be the appropriate substrate for evaluation of biomarkers in dogs with BBB damage from chronic hypothyroidism

• Timing of CSF evaluation may be critical in assessment of biomarkers responsible for vascular pathology of the CNS

Abstract

Chronic canine hypothyroidism is associated with blood–brain barrier (BBB) disruption. We hypothesized that this change is mediated by endothelin-1(ET-1) and matrix metalloproteinases (MMP) -2, -9, and -14, as evidenced by increased concentrations of these proteins in cerebrospinal fluid (CSF) compared to controls.

CSF from 18 dogs, 9 controls and 9 with experimentally induced hypothyroidism was collected before and 6, 12, and 18 months after induction of hypothyroidism. Concentrations of ET-1 using an ELISA kit, and for MMP-2, -9, and -14 using gelatinase zymography were measured in CSF.

ET-1 was undetectable in CSF of control and hypothyroid dogs at all time-points. Constitutively expressed MMP-2 was detectable in CSF samples in all dogs at all time-points. No other MMPs were detectable in CSF.

No differences in CSF concentrations of ET-1 and MMP-2, 9, and 14 were found between hypothyroid and euthyroid dogs. Therefore, ET-1 and MMP-2, 9, and 14 are unlikely to be primary mediators of BBB damage in chronically hypothyroid dogs.

Introduction

Adequate thyroid function is critical for growth and development of animals. In the aging canine, thyroid function has an important role in the maintenance of body system metabolism including the nervous system. The most common clinical manifestations of hypothyroidism are usually dermatologic and systemic and include lethargy, weight gain, alopecia, seborrhea, pyoderma, poor hair coat, and otitis externa (Panciera, 1994). Neurologic signs from chronic hypothyroidism may involve disturbances to either the peripheral nervous system, muscle, or central nervous system. Various case reports exist describing neuromuscular abnormalities associated with hypothyroidism (Indrieri et al., 1987, Budsburg et al., 1993, Jaggy et al., 1994). These reports demonstrate evidence supporting primary myopathic disease, denervation, and demyelination associated with spontaneous hypothyroidism. More recently Utsugi et al. (2014) showed biopsy confirmed pathologic change in a dog with hypothyroidism and clinical signs of polyneuropathy; results demonstrated subperineural edema and myelinated nerve fiber loss consistent with neuronal degeneration and similar to that reported in people with hypothyroid neuropathy (Dyck and Thomas, 2005). Subclinical myopathic changes have been characterized in our laboratory's group of experimentally induced hypothyroid dogs (Rossmeisl et al., 2009, Rossmeisl, 2010) but we were unable to reproduce peripheral nerve dysfunction (Rossmeisl, 2010). Central neurologic dysfunction due to hypothyroidism can be a life threatening complication and includes vestibular dysfunction, cranial neuropathies, and coma (Bichsel et al., 1988, Jaggy et al., 1994, Panciera, 2001, Higgins et al., 2006, Scott-Moncrieff, 2007, Vitale and Olby, 2007, Blois et al., 2008).

The etiology of CNS signs remains elusive. Myxedematous plaques, cerebral acidosis, hypoglycemia, and atherosclerosis are proposed mechanisms (Higgins et al., 2006, Finora and Greco, 2007, Scott-Moncrieff, 2007). Recently, MRI studies have documented vascular accidents and ischemic injury secondary to hypothyroidism (Patterson et al., 1985, Zeiss and Waddle, 1995, Hess et al., 2003, Hillock et al., 2006, Blois et al., 2008) and atherosclerosis has been confirmed in some cases (Patterson et al., 1985, Hess et al., 2003, Blois et al., 2008). However, not all hypothyroid dogs with clinical signs of brain dysfunction have visible lesions, but they do require thyroid supplement for neurological recovery, suggesting a functional problem in some cases (Higgins et al., 2006, Vitale and Olby, 2007).

Our laboratory has previously documented dysfunction of the blood–brain barrier (BBB) in chronically hypothyroid canines as determined by cerebrospinal fluid (CSF) and serum biomarker analysis of glial and endothelial function (Pancotto et al., 2010). It remains to be determined how this disruption occurs. The primary objective of this study was to identify additional biomarkers, specifically of vascular dysfunction, that may lead to BBB degradation. We hypothesized that BBB disruption in hypothyroidism is mediated by endothelin-1 (ET-1) and matrix metalloproteinases (MMPs) as evidenced by increased concentrations of these proteins in CSF.

ET-1 is secreted by the endothelial cells of brain capillaries and by astrocyte foot process of the BBB (Dehouck et al., 1997, Kastner et al., 2005). It is measurable in the CSF of animal models and humans with ischemic brain injury (Bian et al., 1994, Lampl et al., 1997, Volpe and Cosentino, 2000) and experimental ischemic brain damage can be attenuated by administration of endothelin receptor antagonists (Lo et al., 2005). For these reasons and because hypothyroid animals may have MRI-identifiable ischemic brain injury (Patterson et al., 1985, Zeiss and Waddle, 1995, Hess et al., 2003, Hillock et al., 2006, Blois et al., 2008), CSF ET-1 was thought to be a good potential marker of this type of injury.

MMPs are zinc-dependent endopeptidases important to the remodeling of extracellular matrix. MMPs have been measured in CSF of clinically normal dogs (Bergman et al., 2002) as well as dogs with canine distemper virus associated leukoencephalitis (Groters et al., 2005), intervertebral disc disease (Levine et al., 2006), and CNS neoplasia (Turba et al., 2007). In all of the disease states, inflammatory change within the CNS was associated with increased MMP activity and altered BBB permeability. Based on this work MMPs are known to be measurable in CSF and may provide a mechanism for loss of BBB integrity.

Elucidating the mechanisms behind the development of BBB dysfunction will aid our understanding of the critical role of thyroid hormone function in the adult central nervous system. Identifying the primary pathophysiology of BBB damage in this population may lead to specific therapeutic interventions aimed at reversing the underlying change in addition to thyroid hormone replacement therapy for more rapid and complete neurologic recovery.