Paul Smith, Susannah Greenberg, Donald Heath and John Gosney. Department of Pathology, University of Liverpool, Liverpool, UK. Received for publication i6 ...
Br. |. exp. Path. (I987) 68, 25I-258
Effects of glucocorticoids
on
the rabbit carotid body
Paul Smith, Susannah Greenberg, Donald Heath and John Gosney Department of Pathology, University of Liverpool, Liverpool, UK
Received for publication i6 July I986 Accepted for publication 23 November I986
Summary. Weekly intramuscular injections of slow-release, depot methyl prednisolone to I 5 adult Dutch rabbits did not bring about an increase in the volume of their carotid bodies. However, they caused swelling of chief cells with discrete areas of pallor in the cytoplasm. Factors responsible for these changes appear to be increased storage of catecholamines and an increased number of swollen mitochondria. Keywords: carotid body, corticosteroids, electron microscopy, mitochondria, rabbit
We recently carried out a necropsy on a woman of 56 years who had suffered from bronchial asthma all her life and who had been taking prednisolone for 30 years. She became Cushingoid and developed pronounced cutaneous atrophy. At necropsy both the pituitary and the thyroid glands were normal but the adrenals were extremely small and on sectioning had a very narrow atrophic cortex. Although both carotid bodies were of normal weight (right I4.3 mg, left 7.7 mg) (Heath & Smith I985), histological sections showed increased soli-
slow-release, depot-methyl prednisolone (Depo-medrone) were given to IO male and five female young adult Dutch rabbits. Five controls, three male and two female, were studied for comparison. The animals were killed by injection of pentobarbitone sodium (Nembutal) at periods from 34-IO9 days after the first injection, apart from four who died spontaneously during this time. Necropsy was carried out immediately after death. Both adrenal glands and the pituitary were removed so that the effects of the methyl prednisolone could be confirmed. Both carotid bifurcations were dissected dity of the lobules with pronounced vacuolaout in all the animals. The majority were tion of the chief cells. In view of the finding, we thought it would be of interest to carry fixed in formol saline and processed for light out a histological and ultrastructural study microscopy, but the bifurcations from two of the carotid bodies of rabbits treated with test and two control animals were fixed in long-acting methyl prednisolone to see if 2% glutaraldehyde and processed for elecglucocorticoids induce structural changes in tron microscopy. After losses due to morglomic tissue. tality and procedural casualties a total of I 5 carotid bodies from the test rabbits and four from the controls became available for light Materials and methods microscopical analysis. Paraffin sections of the carotid body, 5 gum Weekly intramuscular injections of 5 mg Correspondence: Dr Paul Smith, Department of Pathology, University of Liverpool, P.O. Box I47, Liverpool L69 3BX, UK.
25I
P. Smith et al. 252 in thickness, were cut at intervals of 20 Pum. These sections were used to determine carotid body volume, the percentage area of a section of it occupied by clusters of glomus cells, and the size of the individual chief cells. The methods employed were as follows: Carotid body volume. The area of cross section of the carotid body on each section was measured with a MOP Videoplan image analyser and digitizer tablet. The outline of the carotid body included the perimeter of all glomic tissue including small blood vessels and interlobular connective tissue but excluding large areas of fibrous tissue and major vessels. From these data the volume of each carotid body was estimated by applying Simpson's rule as cited by Aherne and Dunnill (I982). Fig. i. Diagram to illustrate the definition of While shrinkage during the preparation of 'carotid body area' as the area enclosed by the the sections is inevitable, it is none the less discontinuous line. valid to make comparisons between two groups using this method. It is certainly it was realised at the outset that this was very much more accurate than weighing the difficult as the glomic cells collectively form organ at necropsy, particularly in the rabbit something akin to a syncytium. The same which has a small carotid body with ill- sections used to determine glomic areas in sections of carotid body were re-examined defined boundaries. under higher power. On each slide the area of Percentage of section of carotid body occupied by cross section occupied by five cell clusters glomic tissue. From two sections taken was measured with an image analyser as through the centre of the carotid body the described above. The total number of nuclei percentage occupied by glomic tissue, as of chief cells included within these areas was opposed to connective tissue within the also counted. By dividing the area of the cell confines of the carotid body, was measured clusters by the number of their contained by a MOP Videoplan image analyser as in a nuclei an estimate of the cytoplasmic area previous study on the human carotid body per nucleus was derived. (Hurst et al. I985). The outlines of each glomic lobule were traced (Fig. i). Tangents Qualitative. A study was made of the overall were drawn to the outermost lobules and histological picture of the lobules of the joined to represent an artificial but reproduc- carotid body. Particular attention was paid ible carotid body perimeter. The area to the detection of any possible hyperplasia of enclosed by this outline was measured as the sustentacular (type II) cells or the dark 'total carotid body area' (Fig. i). The com- variant of chief (type I) cells. The cytological bined areas occupied by the lobules them- appearances of the chief cells were studied. selves were then expressed as a percentage of Results the carotid body area. Dimensions of individual chief cells. An attempt Adrenals and pituitary glands was made to gain some assessment of the Morphological changes in the adrenals and dimensions of individual chief cells, although pituitary glands of the animals treated with
Effects of steroids on rabbit carotid body
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253
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Fig. 2. Test rabbit. Lobule of glomic tissue in the left carotid body showing increased solidity due to compaction of the swollen chief cells. H & E, x 375.
Fig. 3. Test rabbit. Lobule of glomic tissue in the carotid body showing compaction of the chief cells. Several of them show pallor and swelling with the formation of vacuoles (arrows) H & E, x 625.
corticosteroid confirmed its expected effects upon these organs. The pituitary corticotrophs of the rabbits given corticosteroid contained the eosinophilic inclusions known as Crooke's hyaline. These are characteristic of states in which elevated levels of corticosteroids suppress the function of these cells. The adrenal cortices of these animals were atrophic. The mean adrenal weight was only 66.8 ± I5.5 mg in comparison with 140.4± I4.5 mg in the case of the controls.
no differential proliferation of the dark variant of chief cells. Even in the control rabbits small or minute discrete areas of pallor could be seen in the cytoplasm, commonly applied to the periphery of the nucleus. In the test animals these cytological appearances were more pronounced. The chief cells seemed swollen and pale and the discrete areas of pallor were larger, sometimes equalling up to a third of the diameter of the nucleus. In many lobules the compaction of the swollen cells was such that their outlines were deformed (Fig. 3).
Qualitative light microscopy The lobules of the carotid bodies of steroidtreated rabbits appeared unusually solid. There was a notable lack of any envelope of elongated sustentacular cells around the. clusters of glomic cells which as a result seemed to merge together (Fig. 2). There was
Quantitative light microscopy The volumes of carotid bodies in the test and control animals are shown in Table i. The mean value for the test rabbits was 68.I mM x io6 and that for the controls was
P. Smith et al.
254 Table i. Carotid body volumes
Volume of carotid bodies
(yM3 x I06) Group
n
Test
I0 9
Control*
Range
Mean
39.6-98.o 68.i (22.40) 4I.3-I48.1 77.3 (29I.3)
Figures in parentheses represent ± one standard deviation of the mean. Comparison of means, t = I. I 684, P not significant. n Number of rabbits in which the carotid bodies were measured. Where both carotid bodies were available from a single animal the average of the two was taken as one parameter. * Data taken from three control rabbits plus six rabbits from an earlier experiment (Smith et al. I986) ofwhich five were female.
Table 2. Percentage of area of section of carotid body occupied by glomic tissue Glomic tissue (%) Group
n
Test
7 I6.I-35.3 22.3 (6.34)
Control 4
Range 4. I-I I .0
Mean
5.9 (3.49)
Figures in parentheses represent ± one standard deviation of the mean. Comparison of means, t = 4.7I 74,
P
