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The purpose of the study was to compare radiographic and histopathologic findings …


Biology Articles » Zoology » Zoopathology » Calcification of intervertebral discs in the dachshund: a radiographic and histopathologic study of 20 dogs » Methods

Methods
- Calcification of intervertebral discs in the dachshund: a radiographic and histopathologic study of 20 dogs

The vertebral columns were obtained from 20 dachshunds that were euthanased for reasons unrelated to research at the Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science. The case material, including size and coat varieties, sex, age and reason for euthanasia is presented in Table 1. The age of the dachshunds ranged from ten months to 13 years (mean 5.3 years) and there were twelve (60.0%) females.

Table 1. Case material.

The vertebral columns were separated from the skull, ribs and pelvis by dearticulation. The tail, including the coccygeal vertebrae, was also removed. The spines were freed of muscle, loose connective tissue and ligaments.

Within 24 hours of death, conventional lateral radiographs were taken of the cervical, thoracic, lumbar and sacral vertebral columns [13]. The radiographic equipment was a Philips Medio 50 CP generator and a Super Rotalix 2550 tube. The cassettes were Cawo 18 × 24 cm rectangle x-ray cassettes containing a green-emitting 100 MR (fine) intensifying screen on one side of a Kodak T-Mat L/RA-film. No grid was used and at exposure the specimens were in direct contact with the cassettes. The voltage and milliampere-seconds used depended upon the size of the vertebrae and varied between 50 and 60 kV and 10 and 20 mAs respectively.

At least four exposures were taken of each dog covering the vertebral column from the first cervical (C1) to the first sacral (S1) vertebra. The total number of calcified discs and their location in the vertebral column were recorded according to earlier described methods [13,14]. Three degrees of calcification of the individual discs were noted and defined as follows (Figure 1):

Figure 1 Lateral radiograph from the seventh cervical (C7) to the fifth thoracic (T5) vertebra in a six-year-old, male, smoothcoated dachshund of standard size (Dog no. 10). Three intervertebral discs; T1–2, T3–4 and T4–5, shows a 'severe', 'moderate' or 'slight' degree of calcification (arrows).

SLIGHT:

- one or more calcified bodies with diameter less than 2 mm, or

- calcification of part of the transitional zone between the nucleus pulposus and the annulus fibrosus, or

- indistinct calcification of a larger part of the nucleus pulposus

MODERATE:

- calcified bodies with diameter less than 2 mm and calcification of part of the transitional zone between the nucleus pulposus and the annulus fibrosus, or

- distinct calcification of a larger part of the nucleus pulposus, or

- indistinct calcification of the entire nucleus pulposus

SEVERE:

- distinct calcification of the entire nucleus pulposus

If there was doubt about calcification or the degree of calcification, additional radiographs were taken of the vertebral section of current interest.

Immediately after the radiographic examination, specimens for histological examination were collected by removing each intervertebral disc between the second cervical (C2) and first sacral (S1) vertebra in all of the 20 vertebral columns. With tissue forceps, a scalpel handle and no. 11 blade the discs were carefully separated from the adjacent vertebral body end plates (extremitas cranialis et caudalis) and removed.

All specimens (520 discs) were fixed in 4% phosphate-buffered formaldehyde, pH 7.2. Subsequently the discs were dehydrated in ethanol, equilibrated in xylene and embedded in paraffin. Decalcifications of the specimens were not performed. The discs were sectioned transversely at about 5 μm and stained with hematoxilin-eosin and von Kossa, a method for demonstration of calcium [15]. During the preparation process all sections concerning 17 discs became incomplete and consequently these discs were regarded as withdrawals. By this, 503 discs were available for a final histopathologic examination by light microscopy.

Three degrees of calcification of the individual discs were noted, and for each category, all findings might appear either separately or in combination with other lesions. The degrees of calcification were defined as follows:

SLIGHT (Figure 2):

Figure 2 Histopathology of disc no. 6 (C7–T1) from Dog no. 10. Calcium deposits (arrows) are seen multifocally occupying less than half of the area of the nucleus pulposus (Np): 'slight' degree of calcification (1). Af = annulus fibrosus. Von Kossa; Bar = 1.6 mm.

- single or multiple foci of calcification occupying less than half of the area of the nucleus pulposus

- single or multiple small foci of calcification within the annulus fibrosus

MODERATE (Figure 3):

Figure 3 Histopathology of disc no 10 (T4–5) from Dog no. 10. Calcium deposits (arrows) form a thin discontinuous ring surrounding the nucleus pulposus (Np), with one focus extending into the annulus fibrosus: 'moderate' degree of calcification (2). Af = annulus fibrosus. Von Kossa; Bar = 1.6 mm.

- single or multiple foci of calcification occupying more than half, but not all, of the area of the nucleus pulposus

- single or multiple medium size foci of calcification within the annulus fibrosus

- thin calcified ring, continuous or discontinuous, either surrounding the nucleus pulposus or localised within the annulus fibrosus

SEVERE (Figure 4):

Figure 4 Histopathology of disc no. 7 (T1–2) from Dog no. 10. Calcium deposits (arrows) in the nucleus pulposus (Np) and in a broad discontinuous ring within the annulus fibrosus (Af): 'severe' degree of calcification (3). Von Kossa; Bar = 1.6 mm.

- widespread or total calcification of the nucleus pulposus

- single or multiple large foci of calcification within the annulus fibrosus

- broad calcified ring, continuous or discontinuous, either surrounding the nucleus pulposus or localised within the annulus fibrosus

If two or more sections of the same disc were read to have different degrees of calcification, the section with the most severe degree of calcification determined the evaluation of that disc.

Radiographs and histological sections were evaluated independently.


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