Hypomyelination syndrome of the Weimaraner dog is a disease characterised by a reduction or absence of myelin in the axons of the central nervous system (CNS) exclusively. The objective of this study was to analyse the cause of this deficiency of myelin. Tissue samples of the CNS of three Weimaraner dogs with neurological signs were fixed in 10% formalin and embedded in paraffin wax, and histochemical, immunohistochemical and ultrastructural studies were performed. Histochemical staining with haematoxylin and eosin and Kluver-Barrera techniques showed generalised pallor in the peripheral areas of the ventral and lateral funiculi of the spinal cord. Immunohistochemical analysis showed a weak expression of both proteolipid protein (PLP) and myelin basic protein (MBP) and a marked decrease of Olig2+ cells in the demyelinated areas. The immunohistochemical findings suggested a myelination or remyelination failure because of the smaller population of oligodendrocytes. However, PLP gene mutations may also be the cause of the decrease of PLP expression as described in other species.
Hypomyelination syndrome (HS) is characterised by a reduction or absence of myelin in the axons of the central nervous system (CNS) exclusively. HS has been described in man and in a wide variety of domestic mammals with the exception of horses (Watabe and others 1973, Maxie and Youssef 2007). In animals, the disease has an early onset and a common dominant clinical feature of severe generalised tremors. The syndrome is known as “shaking pup” syndrome, congenital “tremor”, “shaker” or “trembler” (Griffiths and others 1981, Maxie and Youssef 2007). The myelin deficiency may be permanent or simply delayed, and in some cases the clinical signs disappear with age. In most of the cases described in dogs, both brain and spinal cord were affected but HS of variable severity has been described in different breeds (Kornegay and others 1987, Maxie and Youssef 2007). Samoyeds, springer spaniels and Dalmatian had more severe brain and spinal cord lesions than the chow-chow, Bernese mountain dogs and Weimaraner dogs (Kornegay and others 1987, Comont and others 1988).
The cause of HS is not known in all affected species and breeds. In humans, the HS called Pelizaeus-Merzbacher disease is caused by a mutation in the proteolipid protein (PLP) gene that produces a deficiency in PLP (Garbern 2007). In dogs, a genetic basis has been demonstrated in springer spaniels exclusively, where the severe myelin deficiency is caused by the shaker pup (shp) mutation of the myelin PLP gene (Nadon and others 1990). PLP is the main protein of the compact myelin in the CNS, and its function is required for the development and maturation of oligodendrocytes in postnatal stages (Ligon and others 2006). In Weimaraner dogs, a heritable basis has been proposed but not confirmed, and delayed oligodendrocyte differentiation has been suggested as the underlying defect (Maxie and Youssef 2007). The identification of Olig1 and Olig2, basic helix-loop-helix (bHLH) transcription factors that regulate key stages of early oligodendrocyte development, has provided a critical insight into the origin of oligodendrocyte precursor cells, together with their relationship to other CNS lineages (Ligon and others 2006).
The present work describes three cases of HS in Weimaraner dogs with clinical, pathological, histochemical, ultrastructural and immunohistochemical characterisation. The latter study has not been performed in the Weimaraner HS and was aimed to analyse the cause of myelin deficiency.
Case 1. A local practitioner (Córdoba) was consulted by a Weimaraner breeder because all four dogs from a litter had mild (three dogs) to severe (one dog) intentional tremors from three weeks after birth. The latter dog also had continuous head bobbing, and the clinical signs were progressively worsening. This dog was euthanased and used for the case report.
Cases 2 and 3. Two 30-day-old Weimaraner male litter mates were referred to Hospital ARS Veterinaria (Barcelona) with neurological deficiencies since birth that included intentional tremors, marked ataxia, dysmetria and vertical nystagmus. Attitude, postural reactions, vision, and cranial nerve and spinal reflexes were otherwise normal. Because of the severity of symptoms, all three dogs were euthanased, case 1 at 26 days and cases 2 and 3 at 32 days.
At post-mortem examination, no gross lesions were found. Tissue samples were taken from different organs and tissues, including spinal cord, medulla oblongata, cerebellum, pons, mesencephalon, diencephalon, cerebral cortex, cerebral basal nuclei and hippocampus. The samples were fixed in 10% formalin and embedded in paraffin wax. As a control, formalin-fixed, paraffin wax embedded tissue samples of matched areas of the CNS of 28- and 42-day-old crossbreed dogs were retrieved from the Animal Tissue Bank of Catalonia. Formalin-fixed cervical spinal cord sections of all three dogs were also used for electron microscopy examination. The avidin biotin-peroxidase complex (ABC) immunohistochemical method was used to evaluate the expression of myelin basic protein (MBP), PLP, non-phosphorylated (n ph NF) and phosphorylated neurofilament (ph NF) triplet proteins, Olig2, glial fibrillary acidic protein (GFAP), and heat shock protein 25 (HSP25). Further details concerning the antigens, sources, dilutions and antigen retrieval methods are shown in Table 1. Tissue sections of cases and control dogs were treated with either normal rabbit serum (for polyclonal antibodies) or normal mouse serum (IgG1 or IgG2a) (for monoclonal antibodies), and the primary antibodies were omitted to serve as negative technical controls. The incubation of Weimaraner dog skin, nerve and muscle tissue sections with specific primary antibodies served as positive controls. Immunostaining results were evaluated by four people (YM, CC, MP and JMM). The number of MBP, PLP, GFAP, NF, PH NF, Olig2 and HSP25 positive cells was evaluated semi-quantitatively and expressed as the percentage of positive cells out of the total number of cells present in the whole tissue section as follows: (+) means less than 20% staining, (++) represents 21 to 60% and (+++) indicate that more than 60% of cells were positive.
Table 1. Technical details of the immunohistochemical methods used in the study
Monoclonal anti-NF 200 (phos. and non-phos.) antibody produced in mouse (clone N52) (Sigma-Aldrich N0142)
Polyclonal rabbit anti-GFAP (Dako, Code. Z0334)
HIER (citrate buffer pH 6 0·01M)
HSP25 polyclonal antibody (rabbit) (MBL International Corporation SR-801)
Histological studies with haematoxylin and eosin (H&E) and Kluber Barrera (KB) stainings (Fig 1) in cases 2 and 3 showed generalised pallor in white matter of the CNS when compared to controls. Peripheral areas of ventral and lateral funiculi were the more affected areas (Fig 1) while the corpus callosum, crus cerebris and pyramids showed very light pallor. In the peripheral nervous system (PNS) H&E and KB stainings were similar in cases 1, 2 and 3 and controls. Ultrastructurally, many axons were either thinly myelinated or non-myelinated in ventral and lateral funiculi of the white matter of the spinal cord (Fig 2), while dorsal funiculi had compacted and organised myelin sheaths. Further, Schmidt-Lantermann incisures and Nageotte bracelets (paranodes) were dilated (Fig 2).
Both PLP and MBP expressions were weaker in all the three Weimaraner dogs than in control dogs (Table 2). MBP expression was weaker in the dorsal and ventral spinothalamic tracts and the tectospinal tracts of the spinal cord (dogs 1, 2 and 3) (Fig 3), as well as in the corpus callosum and the crus cerebri (dogs 2 and 3). PLP expression was weaker in the dorsal and ventral spinothalamic tracts and the tectospinal tracts of the spinal cord (dogs 1, 2 and 3) (Fig 3), as well as in the corpus callosum, the crus cerebri and the Pyramids (dogs 2 and 3). In these areas, there were no differences in the expression of NF and ph NF between control dogs and the Weimaraner case 1. Similarly, no differences in the presence and morphology of axons were detected between Weimaraner and control dogs with the silver impregnation method of Bielschowsky. GFAP immunostaining revealed some large astrocytes as well as higher cell density in Weimaraner dogs than in comparable areas of control dogs. In these areas (dorsal and ventral spinothalamic tracts and the tectospinal tracts of the spinal cord in dogs 1, 2 and 3, corpus callosum, crus cerebris and pyramids in dogs 2 and 3), lower numbers of cells expressing Olig2 were detected in Weimaraner dogs in comparison with the same areas of the CNS of the control dogs as well as with other areas of their own CNS (Fig 3; Table 2). Glial cells and neurons, especially in the neocortex, cerebellar cortex and spinal cord, showed higher number of HSP25 positive cells in comparison with the same areas in control dogs (Table 2).
Table 2. Semi-quantitative evaluation of MBP, PLP, GFAP, NF, ph NF, Olig2 and HSP25 expression in Weimaraner and control dogs
Spinal cord spinocerebellar tract
C Control dogs, W Weimaraner dogs, ND Not done, HSP25 Heat shock protein 25, ph NF Phosphorylated neurofilament, NF neurofilament
The three new cases of HS shared the clinical, macroscopic, structural and ultrastructural features of the disease previously described in the Weimaraner dogs (Kornegay and others 1987, Comont and others 1988). Myelin deficiency caused a severe tremor syndrome without macroscopic changes in the CNS and microscopic pale staining areas in the white matter of spinal cord (in all cases) and brain (cases 2 and 3). Ultrastructurally, the axons in these areas were either non-myelinated or thinly myelinated and there was evidence of immature myelin as myelin uncompacted sheaths. Leukodystrophies and spongy degeneration are also diseases characterised by abnormalities of central myelinogenesis. The oligodendrocytes are affected directly or indirectly, and this is reflected in the production of CNS myelin of diminished quantity, quality or both. Many of these diseases are inherited and manifested from or shortly after birth (Summers and others 1995). In the investigation of myelin deficiencies the use of both traditional stains for myelin and axons as well as immunohistochemical techniques for specific antigens such as MBP, PLP or Olig2 are helpful to evaluate the white matter. Biochemically, myelin is largely composed of cholesterol, galactocerebroside and phospholipids and, to a lesser extent, proteins. In the CNS PLP is the main protein of the compact myelin while MBP is the main protein of the major dense lines of myelin sheaths (Arroyo and Scherer 2000). In addition, these proteins play an important role in maintaining the stability of myelin sheaths (Maxie and Youssef 2007).
Decreased levels of PLP and MBP expression have been detected immunocytochemically in the spinal cord, brainstem, hemispheres and optic nerve of the springer spaniel and Samoyed HS, as well as murine and feline HSs (Cummings and others 1986). No immunohistochemical studies have been previously applied to study the HS of the Weimaraner breed. Decreased immunohistochemical expression of both PLP and MBP was observed in different areas of the CNS of all the three Weimaraner dogs studied. It has been suggested that PLP may be important in the maturation of oligodendrocytes (Omlin and Anders 1983, Hudson and others 1987, Nadon and others 1990), and hypomyelinogenesis has been associated to X-linked mutations in the PLP gene in human, jimpy mouse and spaniel dogs (Duncan and others 1983, Hudson and others 1987, Nadon and others 1990, Arroyo and Scherer 2000). The lack of differences in the expression of NF and ph NF between control dogs and the Weimaraner case number 1 indicated that neuronal changes or cytoskeleton loss were absent.
Olig2 is a transcription factor belonging to the family of bHLH proteins; it is involved in embryonic differentiation of glial lineages (Marshall and others 2005). In the adult, Olig2 expression participates in the regulation of formation of new oligodendroglial cells (Ligon and others 2006, Menn and others 2006); loss of Oligo2 function delays oligodendrocyte regeneration (Nishiyama 2007). Following demyelination, an increase in Olig2 expression has been detected as the normal Olig2 response in the damaged areas (Talbott and others 2005, Nishiyama and others 2009). The low amount of Olig2+ cells observed in the demyelinated areas of our dogs points to either a myelination or a remyelination failure due to the absence of oligodendrocytes. Similar pathogenic mechanisms have been described in several human demyelinating diseases (congenital and neonatal disorders, multiple sclerosis), where primary defects in myelinating oligodendrocytes have been described, and loss of Olig2 signal has been detected (Ligon and others 2006). HSP25 is a protein of the family of heat shock proteins induced in response to environmental, physical and chemical stresses in some areas of the cerebral and cerebellar cortex and spinal cord, indicating some degree of cellular stress (Birnbaum 1995, Goldbaum and Richter-Landsberg 2001, Beere 2004). The increased HSP25 expression in Weimaraner dogs may reflect the response to environmental and chemical stresses of neurons and glial cells, respectively.
This study demonstrated lower expression of myelin proteins MBP and PLP in hypomyelinated areas and suggests that the hypomyelination is due to the absence of Olig2+ cells. These findings are similar to previous studies in the springer spaniel. Further studies in PLP mutation are necessary to confirm that a similar gene defect reported in the springer spaniel is the cause of the myelinating defect.
The authors thank Professor Lopez Rivero for his anatomical advise. The authors also wish to thank the excellent technical assistance of Lola Pérez and Ester Blasco. Financial support came from research projects BIO-287, AGL2006-09016 & P07-CVI-02559.
Conflict of interest
None of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.